Compositions and methods for fumonisin detoxification

ABSTRACT

Compositions and methods for the complete detoxification of fumonisin and fumonisin degradation products are provided. Particularly, nucleotide sequences corresponding to the detoxification enzymes are provided. The sequences find use in preparing expression cassettes for the transformation of a broad variety of host cells and organisms.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Divisional Application of U.S. patent applicationSer. No. 09/351,224, filed Jul. 12, 1999 now issued as U.S. Pat. No.6,388,171, herein incorporated by reference.

FIELD OF THE INVENTION

The invention relates to compositions and methods for detoxification ordegradation of fumonisin or AP1. The method has broad application inagricultural biotechnology and crop agriculture and in the improvementof food grain quality.

BACKGROUND OF THE INVENTION

Fungal diseases are common problems in crop agriculture. Many strideshave been made against plant diseases as exemplified by the use ofhybrid plants, pesticides, and improved agricultural practices. However,as any grower or home gardener can attest, the problems of fungal plantdisease continue to cause difficulties in plant cultivation. Thus, thereis a continuing need for new methods and materials for solving theproblems caused by fungal diseases of plants.

These problems can be met through a variety of approaches. For example,the infectious organisms can be controlled through the use of agentsthat are selectively biocidal for the pathogens. Another method isinterference with the mechanism by which the pathogen invades the hostcrop plant. Yet another method, in the case of pathogens that cause croplosses, is interference with the mechanism by which the pathogen causesinjury to the host crop plant. In the case of pathogens that producetoxins that are undesirable to mammals or other animals that feed on thecrop plants, interference with toxin production, storage, or activitycan be beneficial.

Since their discovery and structural elucidation in 1988 (Bezuidenhoutet al. (1988) Journal Chem. Soc., Chem. Commun. 1988:743-745),fumonisins have been recognized as a potentially serious problem inmaize-fed livestock. They are linked to several animal toxicosesincluding leukoencephalomalacia (Marasas et al. (1988) Onderstepoort J.Vet. Res. 55:197-204; Wilson et al. (1990) American Association ofVeterinary Laboratory Diagnosticians: Abstracts 33rd Annual Meeting,Denver, Colo., Madison, Wis., USA) and porcine pulmonary edema (Colvinet al. (1992) Mycopathologia 117:79-82). Fumonisins are also suspectedcarcinogens (Geary et al. (1971) Coord. Chem. Rev. 7:81; Gelderblom etal. (1991) Carcinogenesis 12:1247-1251; Gelderblom et al. (1992)Carcinogenesis 13:433-437). Fusarium isolates in section Liseola producefumonisins in culture at levels from 2 to >4000 ppm (Leslie et al.(1992) Phytopathology 82:341-345). Isolates from maize (predominantlymating population A) are among the highest producers of fumonisin(Leslie et al., supra). Fumonisin levels detected in field-grown maizehave fluctuated widely depending on location and growing season, butboth preharvest and postharvest surveys of field maize have indicatedthat the potential for high levels of fumonisins exists (Murphy et al.(1993) J. Agr. Food Chem. 41:263-266). Surveys of food and feed productshave also detected fumonisin (Holcomb et al. (1993) J. Agr. Food Chem.41:764-767; Hopmans et al. (1993) J. Agr. Food Chem. 41:1655-1658);Sydenham et al. (1991) J. Agr. Food Chem. 39:2014-2018). The etiology ofFusarium ear mold is poorly understood, although physical damage to theear and certain environmental conditions can contribute to itsoccurrence (Nelson et al. (1992) Mycopathologia 117:29-36). Fusarium canbe isolated from most field grown maize, even when no visible mold ispresent. The relationship between seedling infection and stalk and eardiseases caused by Fusarium is not clear. Genetic resistance to visiblekernel mold has been identified (Gendloff et al. (1986) Phytopathology76:684-688; Holley et al. (1989) Plant Dis. 73:578-580), but therelationship to visible mold to fumonisin production has yet to beelucidated.

Fumonisins have been shown in in vitro mammalian cell studies to inhibitsphingolipid biosynthesis through inhibition of the enzyme sphingosineN-acetyl transferase, resulting in the accumulation of the precursorsphinganine (Norred et al. (1992) Mycopathologia 117:73-78; Wang et al.(1991) Biol. Chem. 266:14486; Yoo et al. (1992) Toxicol. Appl.Pharmacol. 114:9-15; Nelson et al. (1993) Annu. Rev. Phytpathol.31:233-252). It is likely that inhibition of this pathway accounts forat least some of fumonisin's toxicity, and support for this comes frommeasures of sphinganine:sphingosine ratios in animals fed purifiedfunonisin (Wang et al. (1992) J. Nutr. 122:1706-1716). Fumonisins alsoaffect plant cell growth (Abbas et al. (1992) Weed Technol. 6:548-552;Van Asch et al. (1992) Phytopathology 82:1330-1332; Vesonder et al.(1992) Arch. Environ. Contam. Toxicol. 23:464-467). Kuti et al. (1993)(Abstract, Annual Meeting American Phytopathological Society, Memphis,Tenn.: APS Press) reported on the ability of exogenously addedfumonisins to accelerate disease development and increase sporulation ofFusarium moniliform and F. oxysporum on tomato.

Enzymes that degrade the fungal toxin fumonisin to the compound AP1 havebeen identified in U.S. Patent No. 5,716,820 and pending U.S. patentapplication Ser. Nos. 08/888,949 and 08/888,950, both filed Jul. 7,1997, and hereby incorporated by reference. Plants expressing afumonisin esterase enzyme, infected by fumonisin producing fungus, andtested for fumonisin and AP1 were found to have low levels of fumonisinbut high levels of AP1. AP1 is less toxic than fumonisin to plants andprobably also animals, but contamination with AP1 is still a concern.The best result would be complete detoxification of fumonisin to anon-toxic form. Therefore enzymes capable of degrading AP1 are necessaryfor the further detoxification of fumonisin.

SUMMARY OF THE INVENTION

Compositions and methods for catabolism and detoxification of fumonisinand fumonisin-degradation products as well as fumonisin-related toxinsare provided. In particular, proteins involved in catabolism andtransmembrane transport of fumonisin and fumonisin catabolic productsare provided. Nucleotide sequences corresponding to the proteins arealso included. The compositions are useful in the detoxification anddegradation of fumonisin. The nucleotide sequences can be used inexpression cassettes for transformation of host cells of interest. Thecompositions and methods of the invention are steps in a catabolicpathway for fumonisin. Thus, organisms can be genetically modified toprovide for the catabolism and detoxification of fumonisin andfumonisin-related toxins.

In particular, expression cassettes for expression of the enzymes inplants and other organisms are provided as well as transformed plantsand other host cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 sets forth the proposed pathway for fumonisin degradation byExophiala spinifera.

FIG. 2 schematically illustrates a plasmid vector comprising the genefor one of the fumonisin degradative enzymes of the invention operablylinked to the ubiquitin promoter.

DETAILED DESCRIPTION OF THE INVENTION

The catabolic pathway for detoxification and degradation of fumonisin isprovided. Particularly, enzymes involved in the degradation of fumonisinfrom Exophiala spinifera (American Type Culture Collection Deposit No.74269) and nucleotide sequences encoding such enzymes are disclosed.Such enzymes and nucleotide sequences find use in the breakdown offumonisin and fumonisin-related toxins as well as degradation productsthereof. In this regard, enzymes can be synthesized and utilized or,alternatively, organisms can be transformed with the DNA sequences ofthe invention and used to detoxify fumonisin.

A proposed pathway for the degradation of fumonisin by Exophialaspinifera is provided in FIG. 1. The present invention encompassesenzymes and nucleotide sequences encoding the enzymes involved in thisdegradation pathway for fumonisin. Compositions of the invention includea flavin monooxygenase, an aldehyde dehydrogenase, a permease, and ap-glycoprotein that are involved in the fumonisin degradation pathway.In particular, the present invention provides for isolated nucleic acidmolecules comprising nucleotide sequences encoding the amino acidsequences shown in SEQ ID NOS:3, 5, 8, and 11, or the nucleotidesequences encoding the DNA sequences obtained from the overlappingclones deposited in a bacterial host with the American Type CultureCollection and assigned Accession Number PTA-299. By “DNA sequenceobtained from the overlapping clones” is intended that the DNA sequenceof the fumonisin degrading enzymes can be obtained by sequencing theindividual clones which together comprise the entire fumonisin degradingenzymes. Further provided are polypeptides having an amino acid sequenceencoded by a nucleic acid molecule described herein, for example thoseset forth in SEQ ID NOS:1, 2, 4, 6, 7, 9 and 10, the DNA sequencesobtained from the overlapping clones deposited in a bacterial host withthe American Type Culture Collection and assigned Accession NumberPTA-299, and fragments and variants thereof.

Ten plasmids containing overlapping clones were deposited with theAmerican Type Culture Collection, Manassas, Virginia, and assignedAccession Number PTA-299. The plasmids designated as F_perm3.5 andF_perm4.4 contain common sequences at the regions were they overlap toform the nucleotide sequence encoding a permease. The plasmidsdesignated as F_p-glyco1L4, F_p-glyco5.13, and F_p-glyco6.43 containcommon sequences at the regions were they overlap to form the nucleotidesequence encoding a p-glycoprotein. And the plasmids designated F_Alde1.1, F_Alde2.2, and F_Alde2.5 contain common sequences at the regionswere they overlap to form the nucleotide sequence of an aldehydedehydrogenase. One of skill in the art by sequencing the clones andaligning the overlap may obtain the entire sequence of the permease, thep-glycoprotein, and the aldehyde dehydrogenase.

These deposits will be maintained under the terms of the Budapest Treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thata deposit is required under 35 U.S.C. §112.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. An “isolated” or “purified” nucleic acidmolecule or protein, or biologically active portion thereof, issubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or substantially free of chemicalprecursors or other chemicals when chemically synthesized. Preferably,an “isolated” nucleic acid is free of sequences (preferably proteinencoding sequences) that naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated nucleic acid molecule cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kbof nucleotide sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived. Aprotein that is substantially free of cellular material includespreparations of protein having less than about 30%, 20%, 10%, 5%, (bydry weight) of contaminating protein. When the protein of the inventionor biologically active portion thereof is recombinantly produced,preferably culture medium represents less than about 30%, 20%, 10%, or5% (by dry weight) of chemical precursors or non-protein-of-interestchemicals.

Fragments and variants of the disclosed nucleotide sequences andproteins encoded thereby are also encompassed by the present invention.By “fragment” is intended a portion of the nucleotide sequence or aportion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native protein and hence degradeor catabolize fumonisn. Alternatively, fragments of a nucleotidesequence that are useful as hybridization probes generally do not encodefragment proteins retaining biological activity. Thus, fragments of anucleotide sequence may range from at least about 20 nucleotides, about50 nucleotides, about 100 nucleotides, and up to the full-lengthnucleotide sequence encoding the proteins of the invention.

A fragment of a fumonisin-degrading nucleotide sequence that encodes abiologically active portion of a fumonisin-degrading protein of theinvention will encode at least 15, 25, 30, 50, 100, 150, 200, or 250,300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200 contiguous aminoacids, or up to the total number of amino acids present in a full-lengthfumonisin-degrading protein of the invention (for example, 545, 487,525, 1,263 amino acids for SEQ ID NOS:3, 5, 8 and 11, respectively).Fragments of a fumonisin-degrading nucleotide sequence that are usefulas hybridization probes for PCR primers generally need not encode abiologically active portion of a fumonisin-degrading protein.

Thus, a fragment of a fumonisin-degrading nucleotide sequence may encodea biologically active portion of a fumonisin-degrading protein, or itmay be a fragment that can be used as a hybridization probe or PCRprimer using methods disclosed below. A biologically active portion of afumonisin-degrading protein can be prepared by isolating a portion ofone of the fumonisin-degrading nucleotide sequences of the invention,expressing the encoded portion of the fumonisin-degrading protein (e.g.,by recombinant expression in vitro), and assessing the activity of theencoded portion of the fumonisin-degrading protein. Nucleic acidmolecules that are fragments of a fumonisin-degrading nucleotidesequence comprise at least 16, 20, 50, 75, 100, 150, 200, 250, 300, 350,400, 450, 500, 550, 600, 650, 700, 800, 900, 1,000, 1,100, 1,200, 1,300,or 1,400, 1500, 1, 600, 1,800, 2,000, 2,200, 2,400, 2,600, 2,800, 3,000,3,200, 3,400, 3,600, 3,800, 3,900 nucleotides, or up to the number ofnucleotides present in a full-length fumonisin-degrading nucleotidesequence disclosed herein (for example, 1,691, 1,638, 1,464, 1,764,1,578, 3,999, 3,792 nucleotides for SEQ ID NOS:1, 2, 4, 6, 7, 9, and 10respectively).

By “variants” is intended substantially similar sequences. Fornucleotide sequences, conservative variants include those sequencesthat, because of the degeneracy of the genetic code, encode the aminoacid sequence of one of the fumonisin-degrading polypeptides of theinvention. Naturally occurring allelic variants such as these can beidentified with the use of well-known molecular biology techniques, as,for example, with polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleotide sequences also includesynthetically derived nucleotide sequences, such as those generated, forexample, by using site-directed mutagenesis but which still encode afumonisin-degrading protein of the invention. Generally, nucleotidesequence variants of the invention will have at least 40%, 50%, 60%,70%, generally, 80%, preferably 85%, 90%, up to 95%, 98% sequenceidentity to its respective native nucleotide sequence.

By “variant” protein is intended a protein derived from the nativeprotein by deletion (so-called truncation) or addition of one or moreamino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.

The proteins of the invention may be altered in various ways includingamino acid substitutions, deletions, truncations, and insertions.Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of the fumonisin-degradingproteins can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; U.S.Pat. No. 4,873,192; Walker and Gaastra, eds. (1983) Techniques inMolecular Biology (MacMillan Publishing Company, New York) and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.), herein incorporated by reference. Conservative substitutions,such as exchanging one amino acid with another having similarproperties, may be preferred.

Thus, the genes and nucleotide sequences of the invention include boththe naturally occurring sequences as well as mutant forms. Likewise, theproteins of the invention encompass both naturally occurring proteins aswell as variations and modified forms thereof Such variants willcontinue to possess the desired ability to degrade or catabolizefumonisin. Obviously, the mutations that will be made in the DNAencoding the variant must not place the sequence out of reading frameand preferably will not create complementary regions that could producesecondary mRNA structure. See, EP Patent Application Publication No.75,444.

The deletions, insertions, and substitutions of the protein sequencesencompassed herein are not expected to produce radical changes in thecharacteristics of the protein. However, when it is difficult to predictthe exact effect of the substitution, deletion, or insertion in advanceof doing so, one skilled in the art will appreciate that the effect willbe evaluated by routine screening assays. That is, the activity can beevaluated by a decrease or loss in the toxic activity of fumonisin orAP1.

Variant nucleotide sequences and proteins also encompass sequences andproteins derived from a mutagenic and recombinogenic procedure such asDNA shuffling. With such a procedure, one or more differentfumonisin-degrading coding sequences can be manipulated to create a newfumonisin-catabolizing possessing the desired properties. In thismanner, libraries of recombinant polynucleotides are generated from apopulation of related sequence polynucleotides comprising sequenceregions that have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. For example, using this approach,sequence motifs encoding a domain of interest may be shuffled betweenthe fumonisin-degrading genes of the invention and other knownfumonisin-catabolizing genes to obtain a new gene coding for a proteinwith an improved property of interest, such as an increased K_(m) in thecase of an enzyme. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer (1994) Nature 370:389-391; Crameri et al. (1997)Nature Biotech. 15:436-438; Moore et al. (1997) J. Mol. Biol.272:336-347; Zhang et al. (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri et al. (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

The carboxylesterase and amine oxidase have been previously described inU.S. Pat. No. 5,716,820 and pending U.S. patent application Ser. Nos.08/888,949 and 08/888,950. Such disclosures are herein incorporated byreference. Thus, the sequences of the invention can be used incombination with those previously disclosed or those disclosed inapplication Serial Nos. 09/352,168 and 09/352,159 entitled “AminoPolyolamine Oxidase Polynucleotides and Related Polypeptides and Methodsof Use”, herein incorporated by reference. The enzymes and nucleotidesequences of the present invention provide a means for continuedcatabolism of the fumonisin-degradation products obtained afterdegradation with at least the carboxylesterase and amine oxidase.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of botany, microbiology, tissueculture, molecular biology, chemistry, biochemistry, and recombinant DNAtechnology, which are within the skill of the art. Such techniques areexplained filly in the literature. See, e.g., Langenheim and Thimann,(1982) Botany: Plant Biology and Its Relation to Human Affairs (JohnWiley); Vasil, ed. (1984) Cell Culture and Somatic Cell Genetics ofPlants, Vol. 1; Stanier et al. (1986) The Microbial World (5th ed.,Prentice-Hall); Dhringra and Sinclair (1985) Basic Plant PathologyMethods (CRC Press); Maniatis et al. (1982) Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, New York); Glover, ed. (1985) DNA Cloning, Vols. I and II; Gait,ed. (1984) Oligonucleotide Synthesis; Hames and Higgins, eds. (1984)Nucleic Acid Hybridization; and the series Methods in Enzymology(Colowick and Kaplan, eds., Academic Press, Inc.).

In describing the present invention, the following terms will beemployed, and are intended to be defined as indicated below.

By “microbe” is meant any microorganism (including both eukaryotic andprokaryotic microorganisms), such as fungi, yeast, bacteria,actinomycetes, algae, and protozoa, as well as other unicellularstructures.

A “fumonisin-producing microbe” is any microbe capable of producing themycotoxin fumonisin or analogues thereof. Such microbes are generallymembers of the fungal genus Fusarium, as well as recombinantly derivedorganisms that have been genetically altered to enable them to producefumonisin or analogues thereof.

By “degrading or catabolizing fumonisin” is meant any modification tothe fumonisin or AP1 molecule that causes a decrease or loss in itstoxic activity. Such a change can comprise cleavage of any of thevarious bonds, oxidation, reduction, the addition or deletion of achemical moiety, or any other change that affects the activity of themolecule. In a preferred embodiment, the modification includeshydrolysis of the ester linkage in the molecule as a first step and thenoxidative deamination. Furthermore, chemically altered fumonisin can beisolated from cultures of microbes that produce an enzyme of thisinvention, such as by growing the organisms on media containingradioactively-labeled fumonisin, tracing the label, and isolating thedegraded toxin for further study. The degraded fumonisin can be comparedto the active compound for its phytotoxicity or mammalian toxicity inknown sensitive species, such as porcines and equines. Such toxicityassays are known in the art. For example, in plants a whole leafbioassay can be used in which solutions of the active and inactivecompound are applied to the leaves of sensitive plants. The leaves maybe treated in situ or, alternatively, excised leaves may be used. Therelative toxicity of the compounds can be estimated by grading theensuing damage to the plant tissues and by measuring the size of lesionsformed within a given time period. Other known assays can be performedat the cellular level, employing standard tissue culture methodologies,e.g., using cell suspension cultures.

For purposes of the invention, the fumonisin or fumonisin degradationproducts will be degraded to at least about 50% to about 10% or less ofthe original toxicity, preferably about 30% to about 5% or less, morepreferably about 20% to about 1% or less.

By “fumonisin esterase” is meant any enzyme capable of hydrolysis of theester linkage in fumonisin. Two examples of such enzymes are ESP1 andBEST1 found in U.S. patent application Ser. No. 5,716,820 and pendingU.S. application Ser. Nos. 08/888,949 and 08/888,950, both filed Jul. 7,1997.

By “structurally related mycotoxin” is meant any mycotoxin having achemical structure related to a fumonisin such as fumonisin B1, forexample AAL toxin, fumonisin B2, fumonisin B3, fumonisin B4, fumonisinC1, funonisin A1 and A2, and their analogues, as well as othermycotoxins having similar chemical structures that would be expected tobe detoxified by activity of the fumonisin degradative enzymeselaborated by Exophiala spinifera, American Type Culture CollectionAccession No. 74269, Rhinocladiella atrovirens, American Type CultureCollection Accession No. 74270, or the bacterium of American TypeCulture Collection Accession No. 55552.

By “amplified” is meant the construction of multiple copies of a nucleicacid sequence or multiple copies complementary to the nucleic acidsequence using at least one of the nucleic acid sequences as a template.Amplification systems include the polymerase chain reaction (PCR)system, ligase chain reaction (LCR) system, nucleic acid sequence basedamplification (NASBA, Cangene, Mississauga, Ontario), Q-Beta Replicasesystems, transcription-based amplification system (TAS), and stranddisplacement amplification (SDA). See, e.g., Persing et al., ed. (1993)Diagnostic Molecular Microbiology: Principles and Applications (AmericanSociety for Microbiology, Washington, D.C.). The product ofamplification is termed an amplicon.

By “host cell” is meant a cell that contains a vector and supports thereplication and/or expression of the expression vector. Host cells maybe prokaryotic cells such as E. coli, or eukaryotic cells such as yeast,insect, amphibian, or mammalian cells. Preferably, host cells aremonocotyledonous or dicotyledonous plant cells, including but notlimited to maize, sorghum, sunflower, soybean, wheat, alfalfa, rice,cotton, and tomato. A particularly preferred monocotyledonous host cellis a maize host cell.

The term “hybridization complex” includes reference to a duplex nucleicacid structure formed by two single-stranded nucleic acid sequencesselectively hybridized with each other.

As used herein, “operably linked” includes reference to a functionallinkage between a promoter and a second sequence, wherein the promotersequence initiates and mediates transcription of the DNA sequencecorresponding to the second sequence. Generally, operably linked meansthat the nucleic acid sequences being linked are contiguous and, wherenecessary to join two protein coding regions, contiguous and in the samereading frame.

As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogues thereof thathave the essential nature of a natural ribonucleotide in that theyhybridize, under stringent hybridization conditions, to substantiallythe same nucleotide sequence as naturally occurring nucleotides and/orallow translation into the same amino acid(s) as the naturally occurringnucleotide(s). A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically, or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Exemplary plant promoters include, but are not limited to,those that are obtained from plants, plant viruses, and bacteria thatcomprise genes expressed in plant cells, such as Agrobacterium orRhizobium. Examples are promoters that preferentially initiatetranscription in certain tissues, such as leaves, roots, seeds, fibers,xylem vessels, tracheids, or sclerenchyma. Such promoters are referredto as “tissue preferred”. A “cell type” specific promoter primarilydrives expression in certain cell types in one or more organs, forexample, vascular cells in roots or leaves. An “inducible” promoter is apromoter that is under environmental control. Examples of environmentalconditions that may effect transcription by inducible promoters includeanaerobic conditions or the presence of light. Another type of promoteris a developmentally regulated promoter. For example, a promoter thatdrives expression during pollen development. Tissue-preferred, cell typespecific, developmentally regulated, and inducible promoters constitutethe class of “non-constitutive” promoters. A “constitutive” promoter isa promoter that is active under most environmental conditions.Constitutive promoters are known in the art and include, for example,35S promoter (Meyer et al (1997) J. Gen. Virol. 78:3147-3151);ubiquitin; as well as those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142.

As used herein, “recombinant” includes reference to a cell or vectorthat has been modified by the introduction of a heterologous nucleicacid or that the cell is derived from a cell so modified. Thus, forexample, recombinant cells express genes that are not found in identicalform within the native (nonrecombinant) form of the cell or expressnative genes that are otherwise abnormally expressed, underexpressed, ornot expressed at all as a result of deliberate human intervention. Theterm “recombinant” as used herein does not encompass the alteration ofthe cell or vector by naturally occurring events (e.g., spontaneousmutation, natural transformation/transduction/transposition) such asthose occurring without deliberate human intervention.

As used herein, a “recombinant expression cassette” is a nucleic acidconstruct, generated recombinantly or synthetically, with a series ofspecified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

The nucleotide sequences of the invention can be used to isolatecorresponding sequences from other organisms. In this manner, methodssuch as PCR, hybridization, and the like can be used to identify suchsequences based on their sequence homology to the sequences set forthherein. Sequences isolated based on their sequence identity to theentire fumonisin-degrading sequences set forth herein or to fragmentsthereof are encompassed by the present invention.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook et al. (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.).See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Known methods ofPCR include, but are not limited to, methods using paired primers,nested primers, single specific primers, degenerate primers,gene-specific primers, vector-specific primers, partially-mismatchedprimers, and the like.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the fumonisin-degradingsequences of the invention. Methods for preparation of probes forhybridization and for construction of cDNA and genomic libraries aregenerally known in the art and are disclosed in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.).

For example, the entire fumonisin-degrading sequences disclosed herein,or one or more portions thereof, may be used as a probe capable ofspecifically hybridizing to corresponding fumonisin-degrading sequencesand messenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique amongfumonisin-degrading sequences and are preferably at least about 10nucleotides in length, and most preferably at least about 20 nucleotidesin length. Such probes may be used to amplify correspondingfumonisin-degrading sequences from a chosen organism by PCR. Thistechnique may be used to isolate additional coding sequences from adesired organism or as a diagnostic assay to determine the presence ofcoding sequences in an organism. Hybridization techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. By “stringent conditions” or “stringent hybridizationconditions” is intended conditions under which a probe will hybridize toits target sequence to a detectably greater degree than to othersequences (e.g., at least 2-fold over background). Stringent conditionsare sequence-dependent and will be different in different circumstances.By controlling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of similarity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length,preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1X to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% fornamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5×to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284:T_(m)=81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M isthe molarity of monovalent cations, % GC is the percentage of guanosineand cytosine nucleotides in the DNA, % form is the percentage offormamide in the hybridization solution, and L is the length of thehybrid in base pairs. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of a complementary target sequencehybridizes to a perfectly matched probe. T_(m) is reduced by about 1° C.for each 1% of mismatching; thus, T_(m), hybridization, and/or washconditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point (T_(m))for the specific sequence and its complement at a defined ionic strengthand pH. However, severely stringent conditions can utilize ahybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermalmelting point (T_(m)); moderately stringent conditions can utilize ahybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than thethermal melting point (T_(m)); low stringency conditions can utilize ahybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York); and Ausubel et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See Sambrook et al. (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.).

In general, sequences that encode for a fumonisin-degradative proteinand hybridize to the fumonisin-degrading sequences disclosed herein willbe at least 40% to 50% homologous, about 60% to 70% homologous, and evenabout 80%, 85%, 90%, 95% to 98% homologous or more with the disclosedsequences. That is, the sequence similarity of sequences may range,sharing at least about 40% to 50%, about 60% to 70%, and even about 80%,85%, 90%, 95% to 98% sequence similarity.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith et al. (1981) Adv. Appl. Math.2:482; by the homology alignment algorithm of Needleman et al (1970) J.Mol. Biol. 48:443; by the search for similarity method of Pearson et al(1988) Proc. Natl. Acad. Sci. 85:2444; by computerized implementationsof these algorithms, including, but not limited to: CLUSTAL in thePC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT,BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package,Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA;the CLUSTAL program is well described by Higgins et al. (1988) Gene73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153; Corpet et aL(1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992) ComputerApplications in the Biosciences 8:155-65, and Person et al. (1994) Meth.Mol. Biol. 24:307-331; preferred computer alignment methods also includethe BLASTP, BLASTN, and BLASTX algorithms (see Altschul et al (1990) J.Mol. Biol. 215:403-410). Alignments are performed using the defaultparameters of the above mentioned programs. Alignment is also oftenperformed by inspection and manual alignment.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, preferably at least 80%, more preferably at least90%, and most preferably at least 95%, compared to a reference sequenceusing one of the alignment programs described using standard parameters.One of skill in the art will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning, and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, more preferably atleast 70%, 80%, 90%, and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (T_(m)) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein. Nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is when the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70% sequenceidentity to a reference sequence, preferably 80%, more preferably 85%,most preferably at least 90% or 95% sequence identity to the referencesequence over a specified comparison window. Preferably, optimalalignment is conducted using the homology alignment algorithm ofNeedleman et al. (1970) J. Mol. Biol. 48:443. An indication that twopeptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides that are “substantially similar” share sequencesas noted above except that residue positions that are not identical maydiffer by conservative amino acid changes.

As indicated, the enzymes and nucleotide sequences encoding such enzymesare involved in the degradation of fumonisin and fumonisin-likecompounds. Such enzymes and nucleotide sequences can be utilized aloneor in combination to engineer microbes or other organisms to metabolizefumonisin and resist its toxic effects.

Fumonisin is produced in the intercellular spaces (apoplast) ofFusarium-infected maize cells. Thus, the apoplast is the preferredlocation for esterase and deaminase, flavin amine oxidase and possiblyother catabolic enzymes. It is possible that some fumonisin coulddiffuse or be transported into the maize cells before it is broken downby the apoplastic enzymes and may escape catabolism. Thus, it may bebeneficial to express a fumonisin pump and reroute the fumonisin ordegradation products in such cells. In this manner, any fumonisinentering the cell will be pumped out and reexposed to catabolic enzymes.Similar toxin pumps exist in other toxin-producing fungi that showresistance to toxins or antibiotics. Such a pump useful in the inventionand disclosed herein is a P-glycoprotein homolog.

More complete catabolism of fumonisin in transgenic organisms may beprovided by esterase and deaminase enzymes. Exophiala enzymes that canfurther oxidize fumonisin breakdown-products are not detectedextracellularly. Such enzymes in all likelihood exist in the cytoplasm,where adequate cofactors such as AND⁺ or NADP are found. Thefumonisin-induced metabolite transporter is predicted to providetransport of degradation products into cells where they can be furtherbroken down by other enzymes. In this manner, a permease enzyme may beutilized in a heterologous system to transport either AP1 precursors orfumonisin degradation products into the cytoplasm.

The monooxygenase is expected to result in the oxidation of 2-OP to acompound that lacks a keto group, having instead a terminal aldehydegroup, or possibly a carboxylate group. See, for example, Trudgill etal. (1984) in Microbial Degradation of Organic Compounds, ed. Gibson(Microbiology Series Vol. 13, Marcel Dekker, New York), Chapter 6; andDavey and Trudgill (1977) Eur. J. Biochem. 74:115.

This reaction is due to a type of enzymatic oxidation referred to asBaeyer-Villiger oxidation, in which monooxygen is inserted adjacent to aketo function, resulting in a lactone or ester linkage. The metabolismof trans-cyclohexane-1,2 diol by Acinetobacter provides a model for theactivity of a Baeyer-Villiger monooxygenase on 2-OP. This diol is firstoxidized to ortho hydroxy cyclohexanone and then a monooxygen isinserted between the quinone and hydroxy functions by theBaeyer-Villiger enzyme, cyclohexanone monooxygenase. This intermediatespontaneously rearranges to a linear aldehyde carboxylic acid. Byanalogy, for 2-OP it is predicted oxygen is inserted between carbons 2and 3 followed by spontaneous cleavage to a C22 aldehyde and aceticacid. Further oxidation by an aldehyde dehydrogenase would convert thiscompound to a carboxylic acid; other catabolic products would also bepossible given the high reactivity of the aldehyde group. Additionalsteps include the use of an aldehyde dehydrogenase to result in theoxidation of the aldehyde product of fumonisin to a hydroxy carboxylicacid.

It is recognized that the DNA sequences of the invention can be insertedinto expression cassettes and used to transform a variety of organisms.Enzymes produced recombinantly may be tested for their ability to modifyfumonisin or a fumonisin byproduct using labeled starting material andappropriate buffer and cofactor conditions. For example, to testaldehyde dehydrogenase activity, the aldehyde dehydrogenase produced ina recombinant manner would be incubated with cofactors, NAD+ or NADP,and ¹⁴C-labeled 2-OP for various times and then an aliquot of thereaction mix spotted on TLC. Enzyme activity would be indicated by theappearance of a new radiolabeled spot at a different Rf on the TLCplate.

The sequences of the invention can be introduced into any host organism.The sequences to be introduced may be used in expression cassettes forexpression in the host of interest where expression in the host isnecessary for transcription.

Where expression cassettes are needed, such expression cassettes willcomprise a transcriptional initiation region linked to the codingsequence or antisense sequence of the nucleotide of interest. Such anexpression cassette is provided with a plurality of restriction sitesfor insertion of the sequence to be under the transcriptional regulationof the regulatory regions. The expression cassette may additionallycontain selectable marker genes.

The marker gene confers a selectable phenotype on the transformed cells.Usually, the selectable marker gene will encode antibiotic resistance,with suitable genes including genes coding for resistance to theantibiotic spectinomycin (e.g., the aada gene), the streptomycinphosphotransferase (SPT) gene coding for streptomycin resistance, theneomycin phosphotransferase (NPTII) gene encoding kanamycin or geneticinresistance; the hygromycin phosphotransferase (HPT) gene coding forhygromycin resistance, genes coding for resistance to herbicides whichact to inhibit the action of acetolactate synthase (ALS), in particularthe sulfonylurea-type herbicides (e.g., the acetolactate synthase (ALS)gene containing mutations leading to such resistance in particular theS4 and/or Hra mutations), genes coding for resistance to herbicideswhich act to inhibit action of glutamine synthase, such asphosphinothricin or basta (e.g., the bar gene), or other such genesknown in the art. The bar gene encodes resistance to the herbicidebasta, and the ALS gene encodes resistance to the herbicidechlorsulfuron.

The transcriptional initiation region, the promoter, may be native oranalogous or foreign or heterologous to the host as well as to thecoding sequence. Additionally, the promoter may be the natural sequenceor alternatively a synthetic sequence. By foreign is intended that thetranscriptional initiation region is not found in the native plant intowhich the transcriptional initiation region is introduced. As usedherein a chimeric gene comprises a coding sequence operably linked to atranscription initiation region that is heterologous to the codingsequence.

The transcriptional cassette will include in the 5′-to-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in the host. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source. Foruse in plants or plant cells, convenient termination regions areavailable from the Ti-plasmid of A. tumefaciens, such as the octopinesynthase and nopaline synthase termination regions. See also Guerineauet al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al.(1990) Plant Cell. 2:1261-1272; Munroe et al. (1990) Gene 91:151-158;Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; Joshi et al.(1987) Nucleic Acids Res. 15:9627-9639.

Nucleotide sequences of the invention are provided in expressioncassettes for expression in the host cell of interest. The cassette willinclude 5′ and 3′ regulatory sequences operably linked to the sequenceof interest. The cassette may additionally contain at least oneadditional sequence to be cotransformed into the organism.Alternatively, the additional sequence(s) can be provided on anotherexpression cassette.

Where appropriate, the gene(s) may be optimized for increased expressionin the transformed plant. That is, the genes can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. Methods are available in the art forsynthesizing plant-preferred genes. See, for example, U.S. Pat. Nos.5,380,831, 5,436,391, and Murray et al. (1989) Nucleic Acids Res.17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondaryMRNA structures.

The expression cassettes may additionally contain 5′ leader sequences inthe expression cassette construct. Such leader sequences can act toenhance translation. Translation leaders are known in the art andinclude: picornavirus leaders, for example, EMCV leader(Encephalomyocarditis 5′ noncoding region) (Elroy-Stein et al. (1989)PNAS USA 86:6126-6130); potyvirus leaders, for example, TEV leader(Tobacco Etch Virus) (Allison et al. (1986); MDMV leader (Maize DwarfMosaic Virus); Virology 154:9-20), and human immunoglobulin heavy-chainbinding protein (BiP), (Macejak et al. (1991) Nature 353:90-94);untranslated leader from the coat protein mRNA of alfalfa mosaic virus(AMV RNA 4) (Jobling et al. (1987) Nature 325:622-625); tobacco mosaicvirus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA,ed. Cech (Liss, New York), pp. 237-256); and maize chlorotic mottlevirus leader (MCMV) (Lommel et al. (1991) Virology 81:382-385). Seealso, Della-Cioppa et aL. (1987) Plant Physiol. 84:965-968. Othermethods known to enhance translation can also be utilized, for example,introns, and the like.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

In the same manner, a plant can be transformed with the nucleotidesequences of the invention to provide complete detoxification offumonisin in the transformed plant and plant products. Such plantsinclude, for example, species from the genera Cucurbita, Rosa, Vitis,Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella,Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis,Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea,Avena, Hordeum, Secale, Triticum, Sorghum, Picea, Caco, and Populus.

As used herein, “transgenic plant” includes reference to a plant thatcomprises within its genome a heterologous polynucleotide. Generally,the heterologous polynucleotide is stably integrated within the genomesuch that the polynucleotide is passed on to successive generations. Theheterologous polynucleotide may be integrated into the genome alone oras part of a recombinant expression cassette. “Transgenic” is usedherein to include any cell, cell line, callus, tissue, plant part orplant, the genotype of which has been altered by the presence ofheterologous nucleic acid including those transgenics initially soaltered as well as those created by sexual crosses or asexualpropagation from the initial transgenic. The term “transgenic” as usedherein does not encompass the alteration of the genome (chromosomal orextra-chromosomal) by conventional plant breeding methods or bynaturally occurring events such as random cross-fertilization,nonrecombinant viral infection, nonrecombinant bacterial transformation,nonrecombinant transposition, or spontaneous mutation.

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggset al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606,Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No.5,563,055), direct gene transfer (Paszkowski et al. (1984) EMBO J.3:2717-2722), and ballistic particle acceleration (see, for example,Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) “Direct DNATransfer into Intact Plant Cells via Microprojectile Bombardment,” inPlant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborgand Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)Biotechnology 6:923-926). Also see Weissinger et al. (1988) Ann. Rev.Genet. 22:421-477; Sanford et al. (1987) Particulate Science andTechnology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926(soybean); Finer and McMullen (1991) In vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Tomes,U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and5,324,646; Tomes et al. (1995) “Direct DNA Transfer into Intact PlantCells via Microprojectile Bombardment,” in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin)(maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm etal. (1990) Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren etal. (1984) Nature (London) 311:763-764; Bowen et al., U.S. Pat. No.5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA84:5345-5349 (Liliaceae); De Wet et al. (1985) in The ExperimentalManipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp.197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports12:250-255 and Christou and Ford (1995) Annals ofBotany 75:407-413(rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize viaAgrobacterium tumefaciens); all of which are herein incorporated byreference.

The modified plant may be grown into plants in accordance withconventional ways. See, for example, McCormick et al. (1986) Plant Cell.Reports 5:81-84. These plants may then be grown, and either pollinatedwith the same transformed strain or different strains, and the resultinghybrid having the desired phenotypic characteristic identified. Two ormore generations may be grown to ensure that the subject phenotypiccharacteristic is stably maintained and inherited and then seedsharvested to ensure the desired phenotype or other property has beenachieved.

The degradative enzymes can be fermented in a bacterial host and theresulting bacteria processed and used as a microbial spray. Any suitablemicroorganism can be used for this purpose. See, for example, Gaertneret al. (1993) in Advanced Engineered Pesticides, Kim (Ed.).

The genes of the invention can be introduced into microorganisms thatmultiply on plants (epiphytes) to deliver enzymes to potential targetcrops. Epiphytes can be gram-positive or gram-negative bacteria, forexample.

The microorganisms that have been genetically altered to contain atleast one degradative gene and protein may be used for protectingagricultural crops and products. In one aspect of the invention, whole,i.e., unlysed, cells of the transformed organism are treated withreagents that prolong the activity of the enzyme produced in the cellwhen the cell is applied to the environment of a target plant. Asecretion signal sequence may be used in combination with the gene ofinterest such that the resulting enzyme is secreted outside the hostcell for presentation to the target plant.

Plant signal sequences, including, but not limited to, signal-peptideencoding DNA/RNA sequences which target proteins to the extracellularmatrix of the plant cell (Dratewka-Kos et al., (1989) J. Biol. Chem.264:4896-4900), the Nicotiana plumbaginifolia extension gene (DeLoose,et al. (1991) Gene 99:95-100), signal peptides which target proteins tothe vacuole like the sweet potato sporamin gene (Matsuka et al. (1991)PNAS 88:834) and the barley lectin gene (Wilkins et al. (1990) PlantCell 2:301-313), signal peptides which cause proteins to be secretedsuch as that of PRIb (Lind et al. (1992) Plant Mol. Biol. 18:47-53), orthe barley alpha amylase (BAA) (Rahmatullah et al. (1989) Plant Mol.Biol. 12:119) and hereby incorporated by reference, or from the presentinvention the signal peptide from the ESP1 or BEST1 gene, or signalpeptides which target proteins to the plastids such as that of rapeseedenoyl-Acp reductase (Verwaert et al. (1994) Plant Mol. Biol. 26:189-202)are useful in the invention.

In this manner, at least one of the genes encoding a degradation enzymeof the invention may be introduced via a suitable vector into amicrobial host, and said transformed host applied to the environment orplants or animals. Microorganism hosts that are known to occupy the“phytosphere” (phylloplane, phyllosphere, rhizosphere, and/orrhizoplane) of one or more crops of interest may be selected fortransformation. These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment with the wild-typemicroorganisms, to provide for stable maintenance and expression of thegene expressing the polypeptide pesticide, and, desirably, to providefor improved protection of the enzymes of the invention fromenvironmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes; fungi,particularly yeast, e.g., Saccharomyces, Pichia, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonasfluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli, and Azotobacter vinlandii; and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesrosues, S. odorus, Kluyveromyces veronae, and Aureobasidium pullulans.

Illustrative prokaryotes, both Gram-negative and -positive, includeEnterobacteriaceae, such as Escherichia, Erwinia, Shigella, Salmonella,and Proteus; Bacillaceae; Rhizobiaceae, such as Rhizobium; Spirillaceae,such as photobacterium, Zymomonas, Serratia, Aeromonas, Vibrio,Desulfovibrio, Spirillum; Lactobacillaceae; Pseudomonadaceae, such asPseudomonas and Acetobacter; Azotobacteraceae; and Nitrobacteraceae.Among eukaryotes are fungi, such as Phycomycetes and Ascomycetes, whichincludes yeast, such as Saccharomyces and Schizosaccharomyces; andBasidiomycetes yeast, such as Rhodotorula, Aureobasidium,Sporobolomyces, and the like.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the protein gene intothe host, availability of expression systems, efficiency of expression,stability of the protein in the host, and the presence of auxiliarygenetic capabilities. Other considerations include ease of formulationand handling, economics, storage stability, and the like.

A number of ways are available for introducing a gene expressing thedegradation enzyme into the microorganism host under conditions thatallow for stable maintenance and expression of the gene. For example,expression cassettes can be constructed that include the DNA constructsof interest operably linked with the transcriptional and translationalregulatory signals for expression of the DNA constructs, and a DNAsequence homologous with a sequence in the host organism, wherebyintegration will occur, and/or a replication system that is functionalin the host, whereby integration or stable maintenance will occur.

Transcriptional and translational regulatory signals include but are notlimited to promoter, transcriptional initiation start site, operators,activators, enhancers, other regulatory elements, ribosomal bindingsites, an initiation codon, termination signals, and the like. See, forexample, U.S. Pat. Nos. 5,039,523 and 4,853,331; EPO 0480762A2; Sambrooket al. supra; Maniatis et al., eds. (1982) Molecular Cloning: ALaboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y.); Davis et al., eds. (1980) Advanced Bacterial Genetics (ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.); and the referencescited therein.

It is recognized that the construction of a catabolic pathway in atransformed organism is a complicated feat. Therefore, any means forassembling the enzymes of interest into an organism of interest isencompassed. For example, a single nucleotide sequence encoding all ofthe desired enzymes or multiples thereof may be transformed into thehost organism. When microorganisms are to be applied to the environmentor to a plant, several microorganisms, each transformed with one, two,three, or more nucleotide sequences of the invention, may be utilized.In this manner, all of the enzymes necessary to bring aboutdetoxification of fumonisin and related products may be presented to theenvironment or to the plant by applying a mixture of transformedorganisms or a single organism capable of expressing the entire pathwayor at least expressing enough of the pathway to detoxify fumonisin.

In plants, nucleotide sequences for an enzyme may be transformed into aplant and crossed with plants expressing a different enzyme. In thismanner, progeny can be obtained having the entire sequence or enough ofthe sequence to detoxify fumonisin. Alternatively, a plant can betransformed with nucleotides encoding several enzymes at the same time.In some tissue culture systems it is possible to transform callus withone nucleotide sequence, establish a stable culture line, and thentransform the callus a second time with a second nucleotide sequence.The process may be repeated to introduce additional sequences.

To facilitate the expression of more than one enzyme in a cell, e.g. aplant cell, fusion proteins may be created. Generally, a spacer regionis included between the proteins. The spacer region may comprise acleavage site for cleavage by an endogenous or introduced protease.

The present invention also relates to a method of detoxifying afumonisin or a structurally related mycotoxin with the enzymes fromExophiala spinifera (American Type Culture Collection Accession No.74269), during the processing of grain for animal or human foodconsumption, during the processing of plant material for silage, or infood crops contaminated with a toxin-producing microbe, such as but notlimited to, tomato. Since the atmospheric ammoniation of corn has provento be an ineffective method of detoxification (see Haumann (1995) INFORM6:248-257), such a methodology during processing is particularlycritical where transgenic detoxification is not applicable.

In this embodiment, the fumonisin degradative enzymes found in Exophialaspinifera (American Type Culture Collection Accession No. 74269), arepresented to grain, plant material for silage, or a contaminated foodcrop, or during the processing procedure, at the appropriate stages ofthe procedure and in amounts effective for detoxification of fumonisinsand structurally related mycotoxins. Detoxification by this method canoccur not only during the processing, but also any time prior to orduring the feeding of the grain or plant material to an animal orincorporation of the grain or food crop into a human food product, orbefore or during ingestion of the food crop. The enzymes ormicroorganisms can be introduced during processing in appropriatemanners, for example, as a wash or spray, or in dried or lyophilizedform or powered form, depending upon the nature of the milling processand/or the stage of processing at which the enzymatic treatment iscarried out. See generally, Hoseney, R. C. (1990) Principles of CerealScience and Technology, American Assn. of Cereal Chemists, Inc.(especially Chapters 5, 6 and 7); Jones, J. M. (1992) Food Safety, EaganPress, St. Paul, Minn. (especially Chapters 7 and 9); and Jelen, P.(1985) Introduction to Food Processing, Restan Publ. Co., Reston, Va.Processed grain or silage to be used for animal feed can be treated withan effective amount of the enzymes in the form of an inoculant orprobiotic additive, for example, or in any form recognized by thoseskilled in the art for use in animal feed. The enzymes of the presentinvention are expected to be particularly useful in detoxificationduring processing and/or in animal feed prior to its use, since theenzymes display relatively broad ranges of pH activity. The esterasefrom Exophiala spinifera (American Type Culture Collection Accession No.74269), showed a range of activity from about pH 3 to about pH 6, andthe esterase from the bacterium of the American Type Culture CollectionAccession No. 55552 showed a range of activity from about pH 6 to aboutpH 9 (U.S. Pat. No. 5,716,820, supra). The APAO enzyme from Exophialaspinifera (American Type Culture Collection Accession No. 74269) has apH range of activity from pH 6 to pH 9.

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be both fertilizers or micronutrient donors or otherpreparations that influence plant growth. They can also be selectiveherbicides, insecticides, fungicides, bactericides, nematicides,mollusicides, or mixtures of several of these preparations, if desired,together with further agriculturally acceptable carriers, surfactants,or application-promoting adjuvants customarily employed in the art offormulation. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g., natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders, or fertilizers.

The enzymes can be introduced during processing in appropriate manners,for example as a wash or spray, or in dried or lyophilized form orpowered form, depending upon the nature of the milling process and/orthe stage of processing at which the enzymatic treatmnent is carriedout. See generally, Hoseney (1990) Principles of Cereal Science andTechnology (American Association of Cereal Chemists, Inc.), especiallyChapters 5, 6, and 7; Jones (1992) Food Safety (Eagan Press, St. Paul,Minn.), especially Chapters 7 and 9; and Jelen (1985) Introduction toFood Processing (Restan Publishing Company, Reston, Va.). Processedgrain or silage to be used for animal feed can be treated with aneffective amount of the enzymes in the form of an inoculant or probioticadditive, for example, or in any form recognized by those skilled in theart for use in animal feed. The enzymes of the present invention areexpected to be particularly useful in detoxification during processingand/or in animal feed prior to its use, since the enzymes displayrelatively broad ranges of pH activity. The enzymes from Exophialaspinifera, American Type Culture Collection Accession No. 74269, showeda range of activity for esterase from about pH 3 to about pH 7 (U.S.Pat. No. 5,716,820, supra). The APAO enzyme from Exophiala spinifera,American Type Culture Collection Accession No. 74269, has a pH range ofactivity from pH 6 to pH 9.

In another embodiment, ruminal microorganisms can be geneticallyengineered to contain and express at least one of the fumonisindegradation enzymes of the invention. The genetic engineering ofmicroorganisms is now an art-recognized technique, and ruminalmicroorganisms so engineered can be added to feed in any art-recognizedmanner, for example as a probiotic or inoculant. In addition,microorganisms, plants, or other organisms or their cultured cells invitro capable of functioning as bioreactors can be engineered so as tobe capable of mass producing the degradative enzymes of Exophialaspinifera (American Type Culture Collection Accession No. 74269).

Another embodiment of the present invention is the use of the enzymes ofthe present invention as detection reagents for fumonisins and relatedcompounds. The enzymes of the present invention can be used as detectionreagents because of the high specificity of the esterase and deaminaseenzymes, and the fact that hydrolysis followed by amine oxidation can bemonitored by detection of hydrogen peroxide or ammonia using standardreagents (analogous to a glucose detection assay using glucose oxidase).Hydrogen peroxide is often measured by linking a hydrogenperoxide-dependent peroxidase reaction to a colored or otherwisedetectable peroxidase product (e.g., Demmano et al. (1996) EuropeanJournal of Biochemistry 238(3):785-789). Ammonia can be measured usingion-specific electrodes: Fritsche et al. (1991) Analytica Chimica Acta244(2):179-182; West et al. (1992) Analytical Chemistry 64(5):533-540,and all herein incorporated by reference) or by GC or otherchromatographic method.

For example, recombinant or non-recombinant, active fumonisin esterase,APAO, and proteins of the invention are added in catalytic amounts to asample tube containing an unknown amount of fumonisins (FB1, FB2, FB3,FB4, or partial or complete hydrolysis products of these). The tube isincubated under pH and temperature conditions sufficient to convert anyfumonisin in the sample to AP1, the AP1 to 2-OP, ammonia, and hydrogenperoxide, and to further degradation products. Then suitable reagentsare added for quantification of the hydrogen peroxide or ammonia thatwere generated stoichiometrically from fumonisins. By comparison withcontrol tubes that received no esterase or APAO enzyme, the amount offumonisin present can be calculated in direct molar proportion to thehydrogen peroxide or ammonia detected, relative to a standard curve.

This invention can be better understood by reference to the followingnonlimiting examples. It will be appreciated by those skilled in the artthat other embodiments of the invention may be practiced withoutdeparting from the spirit and the scope of the invention as hereindisclosed and claimed.

EXPERIMENTAL EXAMPLE 1 Fungal and Bacterial Isolates

Exophiala isolates from maize were isolated as described in U.S. Pat.No. 5,716,820 and pending U.S. application Ser. Nos. 08/888,949 and08/888,950, both filed Jul. 7, 1997, and herein incorporated byreference.

Isolation Methods

Direct isolation of black yeasts from seed was accomplished by plating100 microliters of seed wash fluid onto YPD or Sabouraud agar augmentedwith cycloheximide (500 mg/liter) and chloramphenicol (50 mg/liter).Plates were incubated at room temperature for 7-14 days, and individualpigmented colonies that arose were counted and cultured for analysis offumonisin-degrading ability as described above.

Analysis of Fumonisins and Metabolism Products

Analytical thin-layer chromatography was carried out on 100% silanizedC₁₈ silica plates (Sigma #T-7020; 10×10 cm; 0.1 mm thick) by amodification of the published method of Rottinghaus (Rottinghaus et al.(1992) J. Vet. Diagn. Invest. 4:326, and herein incorporated byreference).

To analyze fumonisin esterase activity, sample lanes were pre-wet withmethanol to facilitate sample application. After application of from 0.1to 2 μl of aqueous sample, the plates were air-dried and developed inMeOH:4% KCl (3:2) or MeOH:0.2 M KOH (3:2) and then sprayed successivelywith 0.1 M sodium borate (pH 9.5) and fluorescamine (0.4 mg/ml inacetonitrile). Plates were air-dried and viewed under long-wave UV.

For analysis of APAO activity, an alternative method was used. Equalvolumes of sample and ¹⁴C-AP1 (1 mg/ml, pH 8) substrate were incubatedat room temperature for six days. Analytical thin-layer chromatographywas then carried out on C60 HPK silica gel plates (Whatman #4807-700;10×10 cm; 0.2 mm thick). After application of from 0.1 to 2 μl ofaqueous sample, the plates were air dried and developed inCHCl₃:MeOH:CH₃COOH:H₂O (55:36:8:1). Plates were then air dried andexposed to PhosphorImager screen or autoradiographic film. A StormPhosphorimager was used to scan the image produced on the screen.

Alkaline Hydrolysis of FB1 to AP1

FB1 or crude fumonisin C₈ material was suspended in water at 10-100mg/ml and added to an equal volume of 4 N NaOH in a screw-cap tube. Thetube was sealed and incubated at 60° C. for 1 hr. The hydrolysate wascooled to room temperature and mixed with an equal volume of ethylacetate, centrifuged at 1000 RCF for 5 minute and the organic (upper)layer recovered. The pooled ethyl acetate layers from two successiveextractions were dried under N₂ and resuspended in distilled H₂O. Theresulting material (the aminopentol of FB1 or “AP1”) was analyzed byTLC.

Enzyme Activity of Culture Filtrate and Mycelium

Exophiala spinifera isolate 2141.10 was grown on YPD agar for 1 week,and conidia were harvested, suspended in sterile water, and used at 105conidia per ml to inoculate sterile Fries mineral salts mediumcontaining 1 mg/ml purified FB1 (Sigma Chemical Co.). After 2 weeksincubation at 28° C. in the dark, cultures were filtered through 0.45micron cellulose acetate filters and rinsed with Fries mineral salts.Fungal mycelium was suspended in 15 mL of 0.1% FB1, pH 5.2+1 mM EDTA+3μg/mL Pepstatin A+1.5 μg/mL Leupeptin and disrupted in a Bead Beater™using 0.1 mm beads and one minute pulses, with ice cooling. Hyphalpieces were collected by filtering through Spin X™ (0.22 μm), and bothmycelial supernatant and original culture filtrates were assayed forfumonisin modification by methods outlined above.

Preparation of Crude Culture Filtrate

Agar cultures grown as above were used to inoculate YPD broth cultures(500 ml) in conical flasks at a final concentration of 105 conidia perml culture. Cultures were incubated 5 days at 28° C. without agitationand mycelia harvested by filtration through 0.45 micron filters undervacuum. The filtrate was discarded, and the mycelial mat was washed andresuspended in sterile carbon-free, mineral salts medium (1 g/literNH₃NO₄; 1 g/liter NaH₂PO₄; 0.5 g/liter MgCl₂; 0.1 g/liter NaCl; 0.13g/liter CaCl₂; 0.02 g/liter FeSO₄ 7H₂0, pH 4.5) containing 0.5 mg/mlalkaline hydrolyzed crude FB1. After 3-5 days at 28° C. in the dark withno agitation the cultures were filtered through low protein binding 0.45micron filters to recover the culture filtrate. Phenylmethyl sulfonylfluoride (PMSF) was added to a concentration of 2.5 mM and the culturefiltrate was concentrated using an Amicon™ YM10 membrane in a stirredcell at room temperature and resuspended in 50 mM sodium acetate, pH 5.2containing 10 mM CaCl₂. The crude culture filtrate (approx. 200-foldconcentrated) was stored at −20° C.

To obtain preparative amounts of enzyme-hydrolyzed fumonisin, 10 mg ofFB1 (Sigma) was dissolved in 20 mL of 50 mM sodium acetate at pH 5.2+10mM CaCl₂, and 0.25 mL of 200×concentrated crude culture filtrate of2141.10 was added. The solution was incubated at 37° C. for 14 hours,and then cooled to room temperature. The reaction mixture was brought toapproximately pH 9.5 by addition of 0.4 mL of 4 N KOH, and the mixturewas extracted twice with 10 mL ethyl acetate. The combined organiclayers were dried under LN₂ and resuspended in dH₂O. 2.5 milligrams oforganic extracted material were analyzed by Fast Atom Bombardment (FAB)mass spectrometry. The resulting mass spectrum showed a major ion at M/z(+1)=406 mass units, indicating the major product of enzymatichydrolysis was AP1, which has a calculated molecular weight of 405.

EXAMPLE 2 Preparation of AP1-induced and Non-induced Mycelium

Liquid cultures of Exophiala spinifera isolate 2141.10 were preparedfrom YPD agar plates (Yeast Extract 10 gm, Bacto-Peptone 20 gm, Dextrose0.5 gm, Bacto-Agar 15 gm per liter of water). Aliquots (400-500 uL) of awater suspension of E. spinifera cells from YPD agar were spreaduniformly onto 150×15 mm YPD agar plates with 4 mm sterile glass beads.The plates were incubated at room temperature for 6-7 days. Themycelia/conidia were transferred from the agar plates into Mineral SaltsMedium (MSM) (Na₂HPO₄ 7H₂O 0.2 gm, NH₄Cl 1.0 gm, CaCl₂ 2H₂O 0.01 gm,FeSO₄ 7H₂O 0.02 gm per liter of distilled water, pH 4.5) and centrifugedat 5000×g, 4° C., 20 minutes to pellet the cells. The cell pellet wasrinsed once in 40 mL MSM and recentrifuged. The rinsed cell pellet wasused to inoculate MSM at a 1:19 ratio of packed cells: MSM. The culturewas supplemented with AP1 to a final concentration of 0.5-1.0 mg/ml andincubated at 28° C., 100 rpm, in the dark to induce catabolic enzymes.The supernatants were removed by filtration through 0.45 celluloseacetate. The remaining mycelial mat was washed with sterile MSM and thenfrozen in liquid nitrogen for storage.

EXAMPLE 3 Effect of FB1 and AP1 on Maize Coleoptiles

Maize coleoptiles from 4 day dark-grown germinated maize seeds wereexcised above the growing point and placed in 96-well microliter platesin the presence of 60 microliters of sterile distilled water containingFB1 or AP1 at approximately equimolar concentrations of 1.5, 0.5, 0.15,0.05, 0.015, 0.005, 0.0015, or 0.0005 millimolar, along with watercontrols. After 2 days in the dark at 28° C. the coleoptiles were placedin the light and incubated another 3 days. Injury or lack thereof wasevaluated as follows:

0 .0005 .0015 .005 .015 .05 .15 .5 1.5 mM FB1 − − − − +/− + + + + AP1 −− − − − − − − + + = brown necrotic discoloration of coleoptile − = nosymptoms (same as water control)

The results (see table above) indicate there is at least a 30-folddifference in toxicity between FB1 and AP1 to maize coleoptiles of thisgenotype. This is in general agreement with other studies where thetoxicity of the two compounds was compared for plant tissues. In Lemnatissues, AP1 was approximately 40-fold less toxic (Vesonder et al.(1992) Arch. Environ. Contam. Toxicol. 23:464-467 (1992)). Studies withboth AAL toxin and FB1 in tomato also indicate the hydrolyzed version ofthe molecule is much less toxic (Gilchrist et al. (1992) Mycopathologia117: 57-64). Lamprecht et al. also observed an approximate 100-foldreduction in toxicity to tomato by AP1 versus FB1 (Lamprecht et al.(1994) Phytopathology 84:383-391).

EXAMPLE 4 Effect of FB1 and AP1 on Maize Tissue Cultured Cells (BlackMexican Sweet, BMS)

FB1 or AP1 at various concentrations was added to suspensions of BMScells growing in liquid culture medium in 96-well polystyrene plates.After 1 week the cell density in wells was observed under low powermagnification and growth of toxin-treated wells was compared to controlwells that received water. Growth of BMS cells was significantlyinhibited at 0.4 micromolar FB1, but no inhibition was observed until 40micromolar AP1. This represents an approximate 100-fold difference intoxicity to maize tissue-cultured cells. Similarly Van Asch et al.observed significant inhibition of maize callus grown on solid medium at1.4 micromolar FB1 (Van Asch et al. (1992) Phytopathology 82:1330-1332).AP1 was not tested in that study, however.

EXAMPLE 5

The polynucleotides were identified using a proprietary transcriptimaging method that compares transcript patterns in two samples andallows cloning of differentially expressed fragments. This technologywas developed by CuraGen® (New Haven, Conn.) (see Published PCT PatentApplication No. WO 97/15690, published May 1, 1997, and herebyincorporated by reference). Fluorescently-tagged, PCR amplified cDNAfragments representing expressed transcripts can be visualized as bandsor peaks on a gel tracing, and the cDNA from differentially expressed(induced or suppressed) bands can be recovered from a duplicate gel,cloned, and sequenced. Known cDNAs can be identified without the needfor cloning, by matching the predicted size and partially known sequenceof specific bands on the tracing.

Two RNA samples were obtained from cultures of E. spinifera grown for aspecified period in a mineral salts medium containing either AP1(induced condition) or gamma-aminobutyric acid (ABA; non-inducedcondition) as a sole carbon source. In the induced condition, fumonisinesterase, amine oxidase, enzyme activities are detected, whereas in thenon-induced condition these activities are not detected. The methodsused for induction of and detection of enzyme activity are describedearlier (see Example 2 and Example 5). RNA was extracted from inducedmycelium by Tri-Reagent methods (Molecular Research Center Inc.,Cincinnati, Ohio) only using frozen tissue samples ground with a mortarand pestle 2-fold and up to 79-fold and greater until slushy and addingan additional extraction after the phase separation by extracting theaqueous phase one time with phenol, and two times with aphenol:chloroform:isoamyl alcohol mixture. The RNAs were submitted forCuraGen® transcript imaging to detect cDNA fragments that are inducedspecifically in the presence AP1. In the resulting gel tracing severalbands were found which showed induction of at least 10-fold in AP1-growncells as compared to cells grown in ABA. One set of induced fragmentscan be matched to the fumonisin esterase cDNA. The cloned bands andpossible functions are provided in Table 2. Highly induced bands andtheir likely function are provided in Tables 2 and 3.

TABLE 2 Best BLAST Clone ID Hit BLAST Hit Name, source, size Probfrom-to Function Monooxygenase M1a0-388 A28550 cyclohexanonemonooxygenase, Acinetobacter 1.4e-22 339-414 Baeyer-Villiger oxidationof (flavin monooxygenase or FMO)EC 1.14.13.22 2-OP1 (AP1-N1), utilizingLength = 543 molecular oxygen and reduced NADPH Or NADH Aldehydedehydrogenase (EC 1.) k0n0-235 Y09876 Aldehyde dehydrogenase (Nicotianatabacum); 1.1e-07 152-191 Oxidation of aldehyde passed Length = 542product of FMO to carboxylic acid Permease r0v0-239 S64084 Cholinetransport protein, yeast 9.3e-05 337-397 Transport of 2-OP1 into theLength = 563 cytoplasm r0w0-424 S51169 amino acid transporter AAP4 -Arabidopsis 0.98  8-76 Transport of 2-OP1 into the w0h0-268 thalianacytoplasm len = 466 r0w0-205 P53744 KAPA/DAPA permease, yeast BIO52.1e-07 446-488 Transport of 2-OP1 into the p0t0-308 Length = 561cytoplasm (contig) Transmembrane pump (P-glycoprotein homolog) r0g1-420S20548 Leptomycin resistance protein, pmd1, 1.8e-37 1255-1359Transmembrane pump that Schizosaccharomyces pombe. or removes FB1 fromthe Length =1362 564-668 cytoplasm as a means of protection against itstoxic activity g0s0-142 Leptomycin resistance protein, pmd1, 527-588Transmembrane pump that Schizosaccharomyces pombe. removes FB1 from theLength =1362 cytoplasm as a means of protection against its toxicactivity 10c0-129 Leptomycin resistance protein, pmd1,Schizosaccharomyces pombe. Length =1362 r0s0-180 Leptomycin resistanceprotein, pmd1,  959-1009 Transmembrane pump that Schizosaccharomycespombe. removes FB1 from the Length =1362 cytoplasm as a means ofprotection against its toxic activity r0c0-193 Leptomycin resistanceprotein, pmd1, 885-945 Transmembrane pump that Schizosaccharomycespombe. removes FB1 from the Length =1362 cytoplasm as a means ofprotection against its toxic activity r0s0-330 Leptomycin resistanceprotein, pmd1, 1024-1110 Transmembrane pump that Schizosaccharomycespombe. removes FB1 from the Length =1362 cytoplasm as a means ofprotection against its toxic activity Loc0-129 S20548 Leptomycinresistance protein, pmd1, .0082 949-988 Transmembrane pump thatSchizosaccharomyces pombe. removes FB1 from the Length =1362 cytoplasmas a means of protection against its toxic activity r0h1-262 Leptomycinresistance protein, pmd1, 1135-1218 Transmembrane pump thatSchizosaccharomyces pombe. removes FB1 from the Length =1362 cytoplasmas a means of protection against its toxic activity i0c0-116 e219956 ATPbinding cassette multidrug transporter, 1026-1114 Transmembrane pumpthat Emericella nidulans Length = 1466 removes FB1 from the cytoplasm asa means of protection against its toxic activity

TABLE 3 Cloned Bands Homology, Comments Predicted function PredictedProduct 1. Transmembrane pump (P-glycoprotein homolog) r0g1-420 g0s0-142l0c0-129 r0s0-180 r0c0-193 r0s0-330 r0h1-262 i0c0-116 Homology toLeptomycin resistance protein, Pmd1, Schizosaccharomyces pombe, Length =1362, or other ABC transporter gene family member. { } All 9 bands showhomology to members of the ABC transporter superfamily. #FB1 Pump:Transmembrane pump that removes FB1 from the cytoplasm as a means ofprotection against its toxicity

2. Small Molecule Permease r0v0-239 r0w0-205 p0t0-308.4 r0w0-424?w0h0-268? Homology to choline transport protein, yeast Length = 563 { }Two bands (r0w0-205 and p0t0-308) contig with each other. 2-OP permease:Transport of 2- OP and/or AP1 into the cytoplasm

3. Flavin Monooxygenase (EC 1.14.13.22) m1a0-388 Homology tocyclohexanone monooxygenase., Acinetobacter. Oxidation of ketoneresulting in carbon-carbon bond breakage to form aldehyde. Utilizes NAD+or NADP+ 2-OP monooxygenase: Intracellular oxidation of 2-OP1 to ahydroxy aldehyde (HA-1) plus acetic acid

4. Aldehyde dehydrogenase k0n0-235 Homology to aldehyde dehydrogenase(Nicotiana tabacum); Length = 542 HA-1 dehdyrogenase: Oxidation ofaldehyde product of FMO to a hydroxy carboxylic acid (HCA-1)

Using sequence derived from each clone, a partial cDNA was obtained by3′ and 5′ RACE-PCR (Chenchik et al. (1995) CLONTECHniques X1:5-8);Chenchik et al. (1996) in A Laboratory Guide to RNA: Isolation,Analysis, and Synthesis, ed. Krieg (Wiley-Liss, Inc.), pp. 273-321. ARACE cloning kit from CLONTECH was used to obtain the RACE amplicons.Briefly, poly A+RNA is transcribed to make first strand cDNA using a“lock-docking” poly T, cDNA synthesis primer, the second strand issynthesized, and the Marathon CDNA adaptor is ligated to both ends ofthe ds cDNA. Diluted template is then used with the Marathon adapterprimer and in separate reactions either a 5′ Gene Specific Primer (GSP)or a 3′GSP is used to produce the 3′ or 5′ RACE amplicon. Aftercharacterization of the RACE product(s) and sequencing, full-lengthcDNAs may be generated by 1) end-to-end PCR using distal 5′ and 3′ GSPswith the adapter-ligated ds cDNA as template, or 2) the cloned 5′ and3′-RACE fragments may be digested with a restriction enzyme that cutsuniquely in the region of overlap, and the fragments isolated andligated. Subsequently, the RACE-generated full-length cDNAs from 1) and2) may be cloned into a suitable vector.

EXAMPLE 6 Pichia Expression of Degradative Enzymes

For cloning into Pichia pastoris expression vector, pPicZalphaA,oligonucleotide primers were designed that contain a 22 bp overlap ofthe 5′ end (sense strand) and 3′ end (antisense strand), respectively ofthe open reading frame of the degradative nucleotide of interest,including the stop codon. In addition, each oligo has a 5′ extensionwith digestible restriction sites that allows cloning of the amplifiedinsert in-frame both into EcoRI/Noti digested pPicZalphaA. pPicZalphaAis an E. coli compatible Pichia expression vector containing afunctional yeast alpha-factor secretion signal and peptide processingsites, allowing high efficiency, inducible secretion into the culturemedium of Pichia. After the generation of the 5′ and 3′ RACE products,the resulting band was cloned into EcoRI/NotI digested pPicZalphaAplasmid.

Pichia can be transformed as described in Invitrogen Manual, EasySelect™ Pichia Expression Kit, Version B, #161219, with the enzymepolynucleotide of interest with either an intron (negative control, noexpression) or without an intron (capable of making an active protein).The Pichia culture fluids and pellets are assayed for enzyme activity asdescribed earlier. The six day culture fluids from the same cultures areused to spike with crude fungal enzyme for positive controls.

The sample 50 μl cell pellets are resuspended in 150 μl cold 50 mMNa-phosphate, pH8.0 and divided into two fresh 500 UL tubes. One tube iskept on ice with no treatment, the pellet suspension, and one tube isused for lysis. An equal volume of 0.1 mm zirconia-silica beads is addedto each tube. The tubes are BeadBeat™ for 15 seconds then cooled on ice5 minutes. This is repeated three times. The crude lysate is thentransferred to another tube for assay or lysate suspension. The TLCassays are performed as follows:

1) pellet suspensions (“PELL”); 10 uL

2) lysate suspensions (“LYS”); 10 uL

3) media controls-mixed 5 uL media with 5 uL crude fungal enzyme (ifavailable); 10uL

4) positive control-used crude fungal enzyme undiluted; 10 uL

5) substrate control-used 50 mM Na-phosphate, pH8.0; 10 uL

cofactor (if required) is added to each reaction mixture

incubate 10 uL each sample+10 uL ¹⁴C-substrate (fumonisin, metabolite,or other potential substrate) (1 mg/mL, pH8) at room temperature for 6days

spot 1.0 uL onto C18 and C60 TLC plates

develop C18 plates in MeOH:4% KCl (3:2)

develop C60 plates in CHCl₃:MeOH:CH₃COOH:H₂O (55:36:8:1)

air-dry plates

expose plates to PhosphorScreen 2-3 days

use Storm Phosphorlmager (Molecular Dynamics) to develop images

EXAMPLE 7 Expression of Degradative Enzymes in E. coli

A vector for expressing the enzymes in E. coli is a prokaryoticglutathione S-transferase (GST) fusion vector for inducible, high-levelintracellular expression of genes or gene fragments as fusions withSchistosomajaponicum GST. GST gene fusion vectors include the followingfeatures: a lac promoter for inducible, high-level expression; aninternal lac Iq gene for use in any E. coli host; and the thrombinfactor Xa or PreScission Protease recognition sites for cleaving thedesired protein from the fusion product. The insert of interest iscloned into the 5′ EcoRI site and a 3′ NotI site allowing in-frameexpression of the fusion peptide. The generation of such an insert isdescribed in the previous example.

E. coli is transformed with the vector containing the coding sequencefor the degradative enzyme as described in BRL catalogue, LifeTechnologies, Inc., catalogue; Hanahan (1983) J. Mol. Biol. 166:557;Jessee et al. (1984) J. Focus 6:4; King et al. (1986) Focus 8:1, andhereby incorporated by reference. The transformed E. coli is induced byaddition of IPTG (isopropyl b-D-thiogalactopyranoside). Samples ofsoluble extract and Samples of insoluble inclusion bodies are tested forenzyme activity as described in Example 7.

EXAMPLE 8 Transformation and Regeneration of Transgenic Plants

Immature maize embryos from greenhouse donor plants are bombarded with aplasmid containing the fumonisin-degradation/transporter enzymenucleotide sequences operably linked to a ubiquitin promoter (FIG. 2).This plasmid also contains the selectable marker gene PAT (Wohlleben etal. (1988) Gene 70:25-37) that confers resistance to the herbicideBialaphos. The preferred construct for expression in maize is thenucleotide sequence of the degradative enzyme either fused to the barleyalpha amylase signal sequence or organellar targeting sequence, or leftintact for expression in the cytoplasm transformation is performed asfollows. All media recipes are in the Appendix.

Preparation of Target Tissue

The ears are surface sterilized in 30% Chlorox bleach plus 0.5% Microdetergent for 20 minutes, and rinsed two times with sterile water. Theimmature embryos are excised and placed embryo axis side down (scutellumside up), 25 embryos per plate, on 560Y medium for 4 hours and thenaligned within the 2.5-cm target zone in preparation for bombardment.

Preparation of DNA

A plasmid vector comprising the fumonisin-degradation/transporter enzymeoperably linked to the ubiquitin promoter is made. This plasmid DNA alsocontains a PAT selectable marker. The plasmid is precipitated onto 1.1μm (average diameter) tungsten pellets using a CaCl₂ precipitationprocedure as follows:

100 μl prepared tungsten particles in water

10 μl (1 μg) DNA in TrisEDTA buffer (1 μg total)

100 μl 2.5 M CaCl₂

10 μl 0.1 M spermidine

Each reagent is added sequentially to the tungsten particle suspension,while maintained on the multitube vortexer. The final mixture issonicated briefly and allowed to incubate under constant vortexing for10 minutes. After the precipitation period, the tubes are centrifugedbriefly, liquid removed, washed with 500 ml 100% ethanol, andcentrifuged for 30 seconds. Again the liquid is removed, and 105 μl 100%ethanol is added to the final tungsten particle pellet. For particle gunbombardment, the tungsten/DNA particles are briefly sonicated and 10 μlspotted onto the center of each macrocarrier and allowed to dry about 2minutes before bombardment.

Particle Gun Treatment

The sample plates are bombarded at level #4 in particle gun #HE34-1 or#HE34-2. All samples receive a single shot at 650 PSI, with a total often aliquots taken from each tube of prepared particles/DNA.

Subsequent Treatment

Following bombardment, the embryos are kept on 560Y medium for 2 days,then transferred to 560R selection medium containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, selection-resistant callus clones are transferred to 288Jmedium to initiate plant regeneration. Following somatic embryomaturation (2-4 weeks), well-developed somatic embryos are transferredto medium for germination and transferred to the lighted culture room.Approximately 7-10 days later, developing plantlets are transferred to272V hormone-free medium in tubes for 7-10 days until plantlets are wellestablished. Plants are then transferred to inserts in flats (equivalentto 2.5″ pot) containing potting soil and grown for 1 week in a growthchamber, subsequently grown an additional 1-2 weeks in the greenhouse,then transferred to classic 600 pots (1.6 gallon) and grown to maturity.Plants are monitored for the expression of afumonisin-degrading/transporter protein.

272 V Ingredient Amount Unit D-I H₂O 950.000 Ml MS Salts (GIBCO11117-074) 4.300 G Myo-Inositol 0.100 G MS Vitamins Stock Solution ##5.000 Ml Sucrose 40.000 G Bacto-Agar @ 6.000 G

Directions:

@=Add after bringing up to volume

Dissolve ingredients in polished D-I H₂O in sequence

Adjust to pH 5.6

Bring up to volume with polished D-I H₂O after adjusting pH

Sterilize and cool to 60° C.

##=Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 gof Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-IH₂0 in sequence.

Bring up to volume with polished D-I H₂O . Make in 400 ml portions.Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for onemonth, unless contamination or precipitation occurs, then make freshstock.

Total Volume (L)=1.00

288 J Ingredient Amount Unit D-I H₂O 950.000 Ml MS Salts 4.300 gMyo-Inositol 0.100 g MS Vitamins Stock Solution ## 5.000 ml Zeatin .5mg/ml 1.000 ml Sucrose 60.000 g Gelrite @ 3.000 g Indoleacetic Acid 0.5mg/ml # 2.000 ml 0.1 mM Abscisic Acid 1.000 ml Bialaphos 1 mg/ml # 3.000ml

Directions:

@=Add after bringing up to volume

Dissolve ingredients in polished D-I H₂O in sequence

Adjust to pH 5.6

Bring up to volume with polished D-I H₂O after adjusting pH

Sterilize and cool to 60° C.

Add 3.5g/L of Gelrite for cell biology.

## =Dissolve 0.100 g of Nicotinic Acid; 0.020 g of Thiamine.HCL; 0.100 gof Pyridoxine.HCL; and 0.400 g of Glycine in 875.00 ml of polished D-IH₂O in sequence.

Bring up to volume with polished D-I H₂O. Make in 400 ml portions.Thiamine.HCL & Pyridoxine.HCL are in Dark Desiccator. Store for onemonth, unless contamination or precipitation occurs, then make freshstock.

Total Volume (L)=1.00

560 R Ingredient Amount Unit D-I Water, Filtered 950.000 ml CHU (N6)Basal Salts (SIGMA C-1416) 4.000 g Eriksson's Vitamin Mix (1000XSIGMA-1511 1.000 ml Thiamine.HCL 0.4 mg/ml 1.250 ml Sucrose 30.000 g2,4-D 0.5 mg/ml 4.000 ml Gelrite @ 3.000 g Silver Nitrate 2 mg/ml #0.425 ml Bialaphos 1 mg/ml # 3.000 ml

Directions:

Add after bringing up to volume

#=Add after sterilizing and cooling to temp.

Dissolve ingredients in D-I H₂O in sequence

Adjust to pH 5.8 with KOH

Bring up to volume with D-I H₂O

Sterilize and cool to room temp.

Total Volume (L)=1.00

560 Y Ingredient Amount Unit D-I Water, Filtered 950.000 ml CHU (N6)Basal Salts (SIGMA C-1416) 4.000 g Eriksson's Vitamin Mix (1000XSIGMA-1511 1.000 ml Thiamine.HCL 0.4 mg/ml 1.250 ml Sucrose 120.000 g2,4-D 0.5 mg/ml 2.000 ml L-Proline 2.880 g Gelrite @ 2.000 g SilverNitrate 2 mg/ml # 4.250 ml

Directions:

@=Add after bringing up to volume

#=Add after sterilizing and cooling to temp.

Dissolve ingredients in D-I H₂O in sequence

Adjust to pH 5.8 with KOH

Bring up to volume with D-I H₂O

Sterilize and cool to room temp.

** Autoclave less time because of increased sucrose**

Total Volume (L)=1.00

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

11 1 1691 DNA Exophiala spinifera misc_feature (0)...(0) flavinmonooxygenase with intron 1 atgtcggcca ccagcaactc cagaggcgat tgttccgtcgcatgcgacgc catcatcgtt 60 ggagccggcc tcagcggcat ctctgctgtg tacaaattgcgaaagctcag actcaacgcc 120 aaaatcttcg agggagcccc cgattttggc ggcgtctggcactggaaccg ctaccctggc 180 gctcgtgttg attcggagac gcccttctac caactgaacattcccgaagt atggaaagac 240 tggacctggt cttgccgcta tcctgaccag aaagagttgctgtcatatgt tcaccactgt 300 gacaagatcc ggggcttgag aaaagacgtc tacttcggagctgaggtggt tgatgcgcgg 360 tatgccagag atctgggcac ctggactgtc aagacgtcggctggccatgt tgcgacggca 420 aagtatctca ttctcgctac ggggttgctc cacaggaagcacactcccgc actccccggc 480 ctcgccgatt tcaacgggaa ggtgattcat tcgagtgcctggcacgaaga cttcgacgca 540 gagggccaga gagtcgccgt catcggtgcc ggggccacaagcatccagat tgttcaggag 600 ttggccaaga aggctgacca ggtaaccatg tttatgcgaaggccgagcta ttgtctgccc 660 atgcggcaac gaacgatgga taggaacgaa cagacagcctggaaggccta ctaccccacg 720 ctgtttgaag cgagtcgaaa gtctcggatt ggattcccggtccaggcacc gtcggttggc 780 atctttgaag tcagccccga gcagcgggag gcctatttcgaagagttgtg ggagcgtggg 840 gcctttaatt ttcttgcttg ccagtaccga gaagtcatggttgacaaaaa ggccaaccga 900 ctggtctatg acttctgggc caaaaagact cgatctcgtatcgtcaatcc ggcaaagaga 960 gatctcatgg ctcctctgga gccgccgtac tggttcggtaccaagcgctc cccactggag 1020 agcgactact acgaaatgct ggacaagccg agcgtcgaaattgtgaatct agaacaatcg 1080 cccattgtgg ctgttacaaa gacaggtgtg ctcttgagtgacggcagcaa gagggaatgc 1140 gacacgatcg tgctggcgac gggtttcgac agtttcactggctcgtgagt gtgctcgatc 1200 atggctccga gtccggacgt ttggctgacc ttgaaagattgacacatatg ggcttgaaaa 1260 acaagcacgg agtggacctg aaggaggtgt ggaaagatggcatatctact tatatgggag 1320 tcttctctca tggcttcccc aatgccttct tcgtcgccacggctcaagcc ccgaccgtcc 1380 tttccaacgg cccaacgatc atagaaaccc aagtcgacttgatcgccgat acaattgcaa 1440 agttggaggc cgagcacgcc acgtccgttg aggcgacgaaatcagcacaa gaggcatggt 1500 cgattatgat tgccaagatg aacgagcaca ctctgttccccttgacggat tcgtggtgga 1560 ctggaggcaa catccctggg aaagcaacac gtgctttaaccttcataggc gggattgctc 1620 tctatgagca gatctgtcaa gagaaggtgg ccaattgggatgggtttgat gtgcttcatg 1680 ctccctgcta a 1691 2 1638 DNA Exophialaspinifera misc_feature (0)...(0) flavin monooxygenase, fully spliced 2atgtcggcca ccagcaactc cagaggcgat tgttccgtcg catgcgacgc catcatcgtt 60ggagccggcc tcagcggcat ctctgctgtg tacaaattgc gaaagctcag actcaacgcc 120aaaatcttcg agggagcccc cgattttggc ggcgtctggc actggaaccg ctaccctggc 180gctcgtgttg attcggagac gcccttctac caactgaaca ttcccgaagt atggaaagac 240tggacctggt cttgccgcta tcctgaccag aaagagttgc tgtcatatgt tcaccactgt 300gacaagatcc ggggcttgag aaaagacgtc tacttcggag ctgaggtggt tgatgcgcgg 360tatgccagag atctgggcac ctggactgtc aagacgtcgg ctggccatgt tgcgacggca 420aagtatctca ttctcgctac ggggttgctc cacaggaagc acactcccgc actccccggc 480ctcgccgatt tcaacgggaa ggtgattcat tcgagtgcct ggcacgaaga cttcgacgca 540gagggccaga gagtcgccgt catcggtgcc ggggccacaa gcatccagat tgttcaggag 600ttggccaaga aggctgacca ggtaaccatg tttatgcgaa ggccgagcta ttgtctgccc 660atgcggcaac gaacgatgga taggaacgaa cagacagcct ggaaggccta ctaccccacg 720ctgtttgaag cgagtcgaaa gtctcggatt ggattcccgg tccaggcacc gtcggttggc 780atctttgaag tcagccccga gcagcgggag gcctatttcg aagagttgtg ggagcgtggg 840gcctttaatt ttcttgcttg ccagtaccga gaagtcatgg ttgacaaaaa ggccaaccga 900ctggtctatg acttctgggc caaaaagact cgatctcgta tcgtcaatcc ggcaaagaga 960gatctcatgg ctcctctgga gccgccgtac tggttcggta ccaagcgctc cccactggag 1020agcgactact acgaaatgct ggacaagccg agcgtcgaaa ttgtgaatct agaacaatcg 1080cccattgtgg ctgttacaaa gacaggtgtg ctcttgagtg acggcagcaa gagggaatgc 1140gacacgatcg tgctggcgac gggtttcgac agtttcactg gctcattgac acatatgggc 1200ttgaaaaaca agcacggagt ggacctgaag gaggtgtgga aagatggcat atctacttat 1260atgggagtct tctctcatgg cttccccaat gccttcttcg tcgccacggc tcaagccccg 1320accgtccttt ccaacggccc aacgatcata gaaacccaag tcgacttgat cgccgataca 1380attgcaaagt tggaggccga gcacgccacg tccgttgagg cgacgaaatc agcacaagag 1440gcatggtcga ttatgattgc caagatgaac gagcacactc tgttcccctt gacggattcg 1500tggtggactg gaggcaacat ccctgggaaa gcaacacgtg ctttaacctt cataggcggg 1560attgctctct atgagcagat ctgtcaagag aaggtggcca attgggatgg gtttgatgtg 1620cttcatgctc cctgctaa 1638 3 545 PRT Exophiala spinifera 3 Met Ser Ala ThrSer Asn Ser Arg Gly Asp Cys Ser Val Ala Cys Asp 1 5 10 15 Ala Ile IleVal Gly Ala Gly Leu Ser Gly Ile Ser Ala Val Tyr Lys 20 25 30 Leu Arg LysLeu Arg Leu Asn Ala Lys Ile Phe Glu Gly Ala Pro Asp 35 40 45 Phe Gly GlyVal Trp His Trp Asn Arg Tyr Pro Gly Ala Arg Val Asp 50 55 60 Ser Glu ThrPro Phe Tyr Gln Leu Asn Ile Pro Glu Val Trp Lys Asp 65 70 75 80 Trp ThrTrp Ser Cys Arg Tyr Pro Asp Gln Lys Glu Leu Leu Ser Tyr 85 90 95 Val HisHis Cys Asp Lys Ile Arg Gly Leu Arg Lys Asp Val Tyr Phe 100 105 110 GlyAla Glu Val Val Asp Ala Arg Tyr Ala Arg Asp Leu Gly Thr Trp 115 120 125Thr Val Lys Thr Ser Ala Gly His Val Ala Thr Ala Lys Tyr Leu Ile 130 135140 Leu Ala Thr Gly Leu Leu His Arg Lys His Thr Pro Ala Leu Pro Gly 145150 155 160 Leu Ala Asp Phe Asn Gly Lys Val Ile His Ser Ser Ala Trp HisGlu 165 170 175 Asp Phe Asp Ala Glu Gly Gln Arg Val Ala Val Ile Gly AlaGly Ala 180 185 190 Thr Ser Ile Gln Ile Val Gln Glu Leu Ala Lys Lys AlaAsp Gln Val 195 200 205 Thr Met Phe Met Arg Arg Pro Ser Tyr Cys Leu ProMet Arg Gln Arg 210 215 220 Thr Met Asp Arg Asn Glu Gln Thr Ala Trp LysAla Tyr Tyr Pro Thr 225 230 235 240 Leu Phe Glu Ala Ser Arg Lys Ser ArgIle Gly Phe Pro Val Gln Ala 245 250 255 Pro Ser Val Gly Ile Phe Glu ValSer Pro Glu Gln Arg Glu Ala Tyr 260 265 270 Phe Glu Glu Leu Trp Glu ArgGly Ala Phe Asn Phe Leu Ala Cys Gln 275 280 285 Tyr Arg Glu Val Met ValAsp Lys Lys Ala Asn Arg Leu Val Tyr Asp 290 295 300 Phe Trp Ala Lys LysThr Arg Ser Arg Ile Val Asn Pro Ala Lys Arg 305 310 315 320 Asp Leu MetAla Pro Leu Glu Pro Pro Tyr Trp Phe Gly Thr Lys Arg 325 330 335 Ser ProLeu Glu Ser Asp Tyr Tyr Glu Met Leu Asp Lys Pro Ser Val 340 345 350 GluIle Val Asn Leu Glu Gln Ser Pro Ile Val Ala Val Thr Lys Thr 355 360 365Gly Val Leu Leu Ser Asp Gly Ser Lys Arg Glu Cys Asp Thr Ile Val 370 375380 Leu Ala Thr Gly Phe Asp Ser Phe Thr Gly Ser Leu Thr His Met Gly 385390 395 400 Leu Lys Asn Lys His Gly Val Asp Leu Lys Glu Val Trp Lys AspGly 405 410 415 Ile Ser Thr Tyr Met Gly Val Phe Ser His Gly Phe Pro AsnAla Phe 420 425 430 Phe Val Ala Thr Ala Gln Ala Pro Thr Val Leu Ser AsnGly Pro Thr 435 440 445 Ile Ile Glu Thr Gln Val Asp Leu Ile Ala Asp ThrIle Ala Lys Leu 450 455 460 Glu Ala Glu His Ala Thr Ser Val Glu Ala ThrLys Ser Ala Gln Glu 465 470 475 480 Ala Trp Ser Ile Met Ile Ala Lys MetAsn Glu His Thr Leu Phe Pro 485 490 495 Leu Thr Asp Ser Trp Trp Thr GlyGly Asn Ile Pro Gly Lys Ala Thr 500 505 510 Arg Ala Leu Thr Phe Ile GlyGly Ile Ala Leu Tyr Glu Gln Ile Cys 515 520 525 Gln Glu Lys Val Ala AsnTrp Asp Gly Phe Asp Val Leu His Ala Pro 530 535 540 Cys 545 4 1464 DNAExophiala spinifera misc_feature (0)...(0) aldehyde dehydrogenase, fullyspliced cDNA 4 atggttcttt cgcctgacga atacaagagt gaactcttca tcaacaatgaattcgtctcc 60 tccaaggggt ccgagagatt aacgctcacg aacccgtggg acgaatccaccgttgccact 120 gatgttcacg tggccaacgc ggccgatgtc gacagtgcag tagccgcttcggtgcaggcg 180 gtcaaaaagg gcccatggaa gaagttcaca ggtgcacaac gcgcggcgtgcatgcttaag 240 ttcgcggacc tcgccgagaa gaacgccgag aagctcgctc gtctggagtcgctgcccacc 300 ggtagaccgg tgtcgatgat cactcatttc gacattccaa acatggtctccgtgtttcgc 360 tactatgcag gctgggccga caagatcgcc ggaaagacct ttcccgaggacaacggcaag 420 ccgaattggc gttacgagcc gatgggggtg tgtgctggta ttgccagctggaacgcgact 480 tttctttacg tcggctggaa gatagccccc gccctcgccg ccggctgctccttcatcttc 540 aaagcctcgg agaaatcccc gctgggcgtt ctgggcctcg ctcctctcttcgcagaagcc 600 ggattccctc ctggagtcgt gcagttcctc actggagcac gagtgacgggtgaagcattg 660 gcgtcgcaca tggacattgc gaagatcagc ttcacaagat ctgtcggcggtggccgcgcc 720 gtcaagcaag caacactcaa gtccaacatg aagcgcgtca ctctagaactgggggaaaag 780 ccaaccatcg tcttcaacga agctcctctc gaacggcagt cgggggaatcggcaaaggat 840 ttctcaaaat tcgggcaaat ttgggtcccc ccctcctgtt tgctagtgcaatggggaaat 900 ttagcggaga aattccatgg agtccgtcat ggctcatttg gaggctgtcagagatggctt 960 ggccagaacc cattggaacc caagaggacg catggtccct tcgtcgacaagtcccagtac 1020 gacagagtct tgggtaacat tgacgttggc aaggataccg cgcagctcctcactggcgtt 1080 ggtagaaagg gcgacaaggg attcgcgatt gaaccgacga tatttgtcaatcccaaacca 1140 ggcagcaaaa tttggtttga ggagatcttt ggccccgtct tgtccattaagacgttcaag 1200 acggaagaag aggccattga gattgccaat gacacgactt atgggctagcctcggtcatt 1260 tataccaaat ctctcaacag gggtctccgt gtctcgtcgg cgctcgagaccggtggcgtc 1320 tcgatcaact tcccctttat ccccgagaca caaactccgt ttggcggcatgaaacaatcg 1380 ggctcaggca gagagctagg cgaagaaggg ctcaaggcgt acttggagcccaagaccatt 1440 aatatccacg tcaacataga gtga 1464 5 487 PRT Exophialaspinifera 5 Met Val Leu Ser Pro Asp Glu Tyr Lys Ser Glu Leu Phe Ile AsnAsn 1 5 10 15 Glu Phe Val Ser Ser Lys Gly Ser Glu Arg Leu Thr Leu ThrAsn Pro 20 25 30 Trp Asp Glu Ser Thr Val Ala Thr Asp Val His Val Ala AsnAla Ala 35 40 45 Asp Val Asp Ser Ala Val Ala Ala Ser Val Gln Ala Val LysLys Gly 50 55 60 Pro Trp Lys Lys Phe Thr Gly Ala Gln Arg Ala Ala Cys MetLeu Lys 65 70 75 80 Phe Ala Asp Leu Ala Glu Lys Asn Ala Glu Lys Leu AlaArg Leu Glu 85 90 95 Ser Leu Pro Thr Gly Arg Pro Val Ser Met Ile Thr HisPhe Asp Ile 100 105 110 Pro Asn Met Val Ser Val Phe Arg Tyr Tyr Ala GlyTrp Ala Asp Lys 115 120 125 Ile Ala Gly Lys Thr Phe Pro Glu Asp Asn GlyLys Pro Asn Trp Arg 130 135 140 Tyr Glu Pro Met Gly Val Cys Ala Gly IleAla Ser Trp Asn Ala Thr 145 150 155 160 Phe Leu Tyr Val Gly Trp Lys IleAla Pro Ala Leu Ala Ala Gly Cys 165 170 175 Ser Phe Ile Phe Lys Ala SerGlu Lys Ser Pro Leu Gly Val Leu Gly 180 185 190 Leu Ala Pro Leu Phe AlaGlu Ala Gly Phe Pro Pro Gly Val Val Gln 195 200 205 Phe Leu Thr Gly AlaArg Val Thr Gly Glu Ala Leu Ala Ser His Met 210 215 220 Asp Ile Ala LysIle Ser Phe Thr Arg Ser Val Gly Gly Gly Arg Ala 225 230 235 240 Val LysGln Ala Thr Leu Lys Ser Asn Met Lys Arg Val Thr Leu Glu 245 250 255 LeuGly Glu Lys Pro Thr Ile Val Phe Asn Glu Ala Pro Leu Glu Arg 260 265 270Gln Ser Gly Glu Ser Ala Lys Asp Phe Ser Lys Phe Gly Gln Ile Trp 275 280285 Val Pro Pro Ser Cys Leu Leu Val Gln Trp Gly Asn Leu Ala Glu Lys 290295 300 Phe His Gly Val Arg His Gly Ser Phe Gly Gly Cys Gln Arg Trp Leu305 310 315 320 Gly Gln Asn Pro Leu Glu Pro Lys Arg Thr His Gly Pro PheVal Asp 325 330 335 Lys Ser Gln Tyr Asp Arg Val Leu Gly Asn Ile Asp ValGly Lys Asp 340 345 350 Thr Ala Gln Leu Leu Thr Gly Val Gly Arg Lys GlyAsp Lys Gly Phe 355 360 365 Ala Ile Glu Pro Thr Ile Phe Val Asn Pro LysPro Gly Ser Lys Ile 370 375 380 Trp Phe Glu Glu Ile Phe Gly Pro Val LeuSer Ile Lys Thr Phe Lys 385 390 395 400 Thr Glu Glu Glu Ala Ile Glu IleAla Asn Asp Thr Thr Tyr Gly Leu 405 410 415 Ala Ser Val Ile Tyr Thr LysSer Leu Asn Arg Gly Leu Arg Val Ser 420 425 430 Ser Ala Leu Glu Thr GlyGly Val Ser Ile Asn Phe Pro Phe Ile Pro 435 440 445 Glu Thr Gln Thr ProPhe Gly Gly Met Lys Gln Ser Gly Ser Gly Arg 450 455 460 Glu Leu Gly GluGlu Gly Leu Lys Ala Tyr Leu Glu Pro Lys Thr Ile 465 470 475 480 Asn IleHis Val Asn Ile Glu 485 6 1764 DNA Exophiala spinifera misc_feature(0)...(0) permease, partially spliced cDNA 6 aactatggac tccagaccaagtggatacgg cgagaaaggc gggacaaggc agacaacgaa 60 gaacacagag acggcggcggcaggtggtgc gtccgagtcc ctgaacgttc ctctggagaa 120 gaaacaattt ggcaccatcaccatcgtgtc cttggccttt gtgatttgca acagttgggc 180 tggtatctca ggcagtctccagctcgccct actagcgggg gggcccgtca ctctccttta 240 cggcatccta atcagtactctcgtctacat ctgcatcgct ttctcattag ccgaactgac 300 cagcgtctac ccgactgccggtggccaata tcattttgcg tcgatcctgg caccaaaatc 360 aatcaatcgg agcatttcatacgtgtgcgg actcgtgtcg ttgctttcat ggatcgctat 420 cggaagctca gtgaccatgatacctgctca acagatcccg gcgctgatag ccgcctatag 480 tcacacatac tcccaggattcgtggcatgt cttcctcatc tacgagggag tcgcgctggt 540 ggtgctcttg ttcaacttgtttgccctgaa aagaaaccct tgggttcatg aaatcggatt 600 cggcctcacg atcgctctcttcgtgatctc ctttatcgcc attctagcgc ggtccaaccc 660 caaggctcca aactcacaggtatggactgc ttggagcaac tatactggct ggtccgacgg 720 cgtctgcttc atcctgggcctttcgacatc ctgcttcatg ttcattggct tggacgcagc 780 aatgcatctg gctgaagaatgcacagatgc tgctcgtacg gtacccaaag cagtggtcag 840 tgcaatcata attggcttctgcaccgcctt tccatataca atcgcagttc tgtatggaat 900 tacagatctc gactctattctaagttccgc cggctatatt ccattcgaga caatgacgca 960 gtcccttcgg tcgctcagttttgcaacggt cctctcatgt ggcggtatcg tgatggcctt 1020 cttcgccctc aacgctgtacaagagactgc gtctcgactc acctggagct ttgcccggga 1080 caatgggctg gtattttccactcatctcga acgcattcat ccccgctggc aagttcctgt 1140 ttggtctcta ttcgcgacctggggaattct ggccacatgc ggatgtatat ttctaggttc 1200 tagcacagct ttcaatgccttggtcaattc cgccgttgta ctccagcaac tctccttcct 1260 gatcccaatc gccctactcctctaccaaaa gcgagatcca aagttcttgc cgagcactcg 1320 tgcttttgtg ttaccgcgtggaatcgggtt tctggtcaat gtgctagcgg tggtcttcac 1380 gtccgtcacc actgtgtttttcagcttccc actgaccgtg cctacggccg cgtcaaccat 1440 gaattacaca agtgcgattataggcgttgc acttgctctt ggtgtcttga actgggtcgt 1500 gcatgccagg aagcattatcagggacccca cttggagctt gacggacggg tcgtcggagc 1560 agaatttcaa gttgggccatgaattggacg aaatggagac gcgtgtgcaa tgtcaaaaat 1620 tgctggggtg gtactgagagtctggattag ctgcaacgcg ggacaaccga gggtagaaca 1680 ctctgcaatc gagcaggacaatatcaatta ggcaachasv caaaaaaaaa aaaaaaaaaa 1740 aaaaaagcgg ccgctgaattctag 1764 7 1578 DNA Exophiala spinifera misc_feature (0)...(0)permease, fully spliced cDNA 7 atggactcca gaccaagtgg atacggcgagaaaggcggga caaggcagac aacgaagaac 60 acagagacgg cggcggcagg tggtgcgtccgagtccctga acgttcctct ggagaagaaa 120 caatttggca ccatcaccat cgtgtccttggcctttgtga tttgcaacag ttgggctggt 180 atctcaggca gtctccagct cgccctactagcgggggggc ccgtcactct cctttacggc 240 atcctaatca gtactctcgt ctacatctgcatcgctttct cattagccga actgaccagc 300 gtctacccga ctgccggtgg ccaatatcattttgcgtcga tcctggcacc aaaatcaatc 360 aatcggagca tttcatacgt gtgcggactcgtgtcgttgc tttcatggat cgctatcgga 420 agctcagtga ccatgatacc tgctcaacagatcccggcgc tgatagccgc ctatagtcac 480 acatactccc aggattcgtg gcatgtcttcctcatctacg agggagtcgc gctggtggtg 540 ctcttgttca acttgtttgc cctgaaaagaaacccttggg ttcatgaaat cggattcggc 600 ctcacgatcg ctctcttcgt gatctcctttatcgccattc tagcgcggtc caaccccaag 660 gctccaaact cacaggtatg gactgcttggagcaactata ctggctggtc cgacggcgtc 720 tgcttcatcc tgggcctttc gacatcctgcttcatgttca ttggcttgga cgcagcaatg 780 catctggctg aagaatgcac agatgctgctcgtacggtac ccaaagcagt ggtcagtgca 840 atcataattg gcttctgcac cgcctttccatatacaatcg cagttctgta tggaattaca 900 gatctcgact ctattctaag ttccgccggctatattccat tcgagacaat gacgcagtcc 960 cttcggtcgc tcagttttgc aacggtcctctcatgtggcg gtatcgtgat ggccttcttc 1020 gccctcaacg ctgtacaaga gactgcgtctcgactcacct ggagctttgc ccgggacaat 1080 gggctggtat tttccactca tctcgaacgcattcatcccc gctggcaagt tcctgtttgg 1140 tctctattcg cgacctgggg aattctggccacatgcggat gtatatttct aggttctagc 1200 acagctttca atgccttggt caattccgccgttgtactcc agcaactctc cttcctgatc 1260 ccaatcgccc tactcctcta ccaaaagcgagatccaaagt tcttgccgag cactcgtgct 1320 tttgtgttac cgcgtggaat cgggtttctggtcaatgtgc tagcggtggt cttcacgtcc 1380 gtcaccactg tgtttttcag cttcccactgaccgtgccta cggccgcgtc aaccatgaat 1440 tacacaagtg cgattatagg cgttgcacttgctcttggtg tcttgaactg ggtcgtgcat 1500 gccaggaagc attatcaggg accccacttggagcttgacg gacgggtcgt cggagcagaa 1560 tttcaagttg ggccatga 1578 8 525 PRTExophiala spinifera 8 Met Asp Ser Arg Pro Ser Gly Tyr Gly Glu Lys GlyGly Thr Arg Gln 1 5 10 15 Thr Thr Lys Asn Thr Glu Thr Ala Ala Ala GlyGly Ala Ser Glu Ser 20 25 30 Leu Asn Val Pro Leu Glu Lys Lys Gln Phe GlyThr Ile Thr Ile Val 35 40 45 Ser Leu Ala Phe Val Ile Cys Asn Ser Trp AlaGly Ile Ser Gly Ser 50 55 60 Leu Gln Leu Ala Leu Leu Ala Gly Gly Pro ValThr Leu Leu Tyr Gly 65 70 75 80 Ile Leu Ile Ser Thr Leu Val Tyr Ile CysIle Ala Phe Ser Leu Ala 85 90 95 Glu Leu Thr Ser Val Tyr Pro Thr Ala GlyGly Gln Tyr His Phe Ala 100 105 110 Ser Ile Leu Ala Pro Lys Ser Ile AsnArg Ser Ile Ser Tyr Val Cys 115 120 125 Gly Leu Val Ser Leu Leu Ser TrpIle Ala Ile Gly Ser Ser Val Thr 130 135 140 Met Ile Pro Ala Gln Gln IlePro Ala Leu Ile Ala Ala Tyr Ser His 145 150 155 160 Thr Tyr Ser Gln AspSer Trp His Val Phe Leu Ile Tyr Glu Gly Val 165 170 175 Ala Leu Val ValLeu Leu Phe Asn Leu Phe Ala Leu Lys Arg Asn Pro 180 185 190 Trp Val HisGlu Ile Gly Phe Gly Leu Thr Ile Ala Leu Phe Val Ile 195 200 205 Ser PheIle Ala Ile Leu Ala Arg Ser Asn Pro Lys Ala Pro Asn Ser 210 215 220 GlnVal Trp Thr Ala Trp Ser Asn Tyr Thr Gly Trp Ser Asp Gly Val 225 230 235240 Cys Phe Ile Leu Gly Leu Ser Thr Ser Cys Phe Met Phe Ile Gly Leu 245250 255 Asp Ala Ala Met His Leu Ala Glu Glu Cys Thr Asp Ala Ala Arg Thr260 265 270 Val Pro Lys Ala Val Val Ser Ala Ile Ile Ile Gly Phe Cys ThrAla 275 280 285 Phe Pro Tyr Thr Ile Ala Val Leu Tyr Gly Ile Thr Asp LeuAsp Ser 290 295 300 Ile Leu Ser Ser Ala Gly Tyr Ile Pro Phe Glu Thr MetThr Gln Ser 305 310 315 320 Leu Arg Ser Leu Ser Phe Ala Thr Val Leu SerCys Gly Gly Ile Val 325 330 335 Met Ala Phe Phe Ala Leu Asn Ala Val GlnGlu Thr Ala Ser Arg Leu 340 345 350 Thr Trp Ser Phe Ala Arg Asp Asn GlyLeu Val Phe Ser Thr His Leu 355 360 365 Glu Arg Ile His Pro Arg Trp GlnVal Pro Val Trp Ser Leu Phe Ala 370 375 380 Thr Trp Gly Ile Leu Ala ThrCys Gly Cys Ile Phe Leu Gly Ser Ser 385 390 395 400 Thr Ala Phe Asn AlaLeu Val Asn Ser Ala Val Val Leu Gln Gln Leu 405 410 415 Ser Phe Leu IlePro Ile Ala Leu Leu Leu Tyr Gln Lys Arg Asp Pro 420 425 430 Lys Phe LeuPro Ser Thr Arg Ala Phe Val Leu Pro Arg Gly Ile Gly 435 440 445 Phe LeuVal Asn Val Leu Ala Val Val Phe Thr Ser Val Thr Thr Val 450 455 460 PhePhe Ser Phe Pro Leu Thr Val Pro Thr Ala Ala Ser Thr Met Asn 465 470 475480 Tyr Thr Ser Ala Ile Ile Gly Val Ala Leu Ala Leu Gly Val Leu Asn 485490 495 Trp Val Val His Ala Arg Lys His Tyr Gln Gly Pro His Leu Glu Leu500 505 510 Asp Gly Arg Val Val Gly Ala Glu Phe Gln Val Gly Pro 515 520525 9 3999 DNA Exophiala spinifera misc_feature (0)...(0)p-glycoprotein, with introns 9 tatttsccat ctmckatgaa tggcagatgaatcggagaaa cctcgaccaa accaagatgg 60 cagtgagtcg tcctcacacc ctcccccagaaaaggaaacc gaaggcagta tttcagacta 120 tctacgaatc ttcagatatg ccgacaaatacgactggact ctcaatgtca tcgcgctcat 180 ctgcgccatc ggatccgggg cttcccttcctctgatgtcg atcatcttcg gtagcttcac 240 caacaagttc aacaattaca attcgggcgacgggagtcct gaagcgttca aggccgatgt 300 ggatcatttc gtcctgtggt tcgtctacctctttattggg aagtttgtcc tcacgtacgt 360 ttccacggct gccattacca tttcagctatacgaaccact cgaactcttc gacgagtgtt 420 ccttgaatgc accttgcggc aagaggtctggcatttcgac aagcagagca atggagcaat 480 cgccactcag gtcactacca atggcaaccgtatacaaaca ggtattgccg agaaattggt 540 ctttaccgtg caggcacttt caatgttcttttctgcattt gtggtcgctt tggcgtctca 600 gtggaagcta gctttaatca ccatgtccgtcatccctgcc attttcctgg tcaccggcat 660 ctgcatagca attgatgccg ctcaggaggccaggatcacc aggatctact cacgcgccgc 720 tgtcctcgca gaagaagtct tatcatccatccggacagtc catgctttct acgcccagaa 780 gaaaatggtc gaaaaatatg atgtctttttgcagcaagca caccaagaag ggaagaagaa 840 atcgccaaat tatggggtct tgttctcaactgagtacttt tgcatttacg ctgctatcgc 900 actgggcctt ttgggaaagg tttttcgcatgtatcagaat ggcgaggttg ccgacgttgg 960 caaagtcttt actgttgcct ttccgtcacctttagcagcc acgtccatct caatgcttgc 1020 gccttcaggt tcagtcgttt accaacgccgcatcttcggc ctccgaatta ttcagtatca 1080 ttgacaaacc cacgcagctc gacccttctcgacccttttt ggaaagcagc cagagggctg 1140 cttaggtcaa attgagatcc aaaacctggcatttgcctac ccctcccgac catctgccca 1200 agtacttcga gatttcaact tgacaattccagctggcaag acgacggccc tcgtcggtgc 1260 atcaggtagc ggcaaaagca caatggtcggcttacttgaa cggtggtatc tgcccagttc 1320 ggggaggata ttacttgatg ggttggaactgggacaatac aatgtgaaat ggctgagaag 1380 ccgcattcgc ctcgttcaac aggaacctgtgttgtttcgt ggcacaatct tccagaacat 1440 tgccaacggt ttcatggatg agcaacgagatctgcctcgc gaaaaacaaa tggagcttgt 1500 gcaaaaagct tgcaaagcag caatgccgacgtgttcatta atgagcttcc gaacggttat 1560 gagactgaag ttggcgagcg agccggagccttgagtggag gtcaacaagc cgaattgcaa 1620 tcgcacgaag tatcatatcg gatcccaagatcctgttact cgatgaagct accagcgccc 1680 ttgacccgaa ggcggagaaa gtggtccaggaggccttgaa ccgagtgtcc aaagaccgca 1740 ctactttggt cattgcccac aaactagccactgtcatacg actcactatt agggcgaatt 1800 gggccctcta gatgcatgct cgagcggccgccagtgtgac gaattgatgc agaattcggc 1860 ttgtcattac gccgcactgg tgcgtgcacaggacctcggg gctgacgaac aagaagaaca 1920 tgagaagacc ctgcacgaaa aggcagcacgagaagctgct ggtgaacgac cggcacttga 1980 gcgcactcac accactgcca catctcaagctggagacctg gagaagcgga aggtgccggt 2040 cgggactttg ggctactcgc tcctaaaatgcatcctaatc atgttctacg aacaaaaaaa 2100 tctctactgg tgcttcttgt tgtcaacaatagcggttctg atatgcgcgg ccacatttcc 2160 aggacaagcc cttttgtttt cgagattgctcactgtcttc gagttgagtg gtcatgcggc 2220 acaggaacgg gcagactttt atagtctgatgttctttgtc gtggctctag gaaatctagt 2280 aggatatttc acgattggct ggacatgcaacgttgtttca caagttgtca cccatcgcta 2340 tcgagccgaa atgttccaac gagtactggatcaagacatc gaattcttcg acatcccgga 2400 gaatacttct ggtgctctca catcgcaactgtcagctcta cccacgcagt tgcaggagtt 2460 gatatcaaca aattcttctc atttttatcgttgtcgtaca acatcctctc gagcagtgct 2520 ctagcactag cctatggatg gaaactgggcctggtggttg tgtttggtgc acttccaccc 2580 ctgcttttgg ctggctacct cagaattcgtcttgagacga agctagaagc cggaaactcg 2640 gcaaactttg cagaaagtgc tgggcttgcaagcgaagcag ttaccgcgat ccggaccgtc 2700 tcatctttga ctctcgaagg scatgttctccaacagtact cggacatgtt gagcaaggtc 2760 gtgctaagat catccaaagc tttggtttggacgatgtttt ggttctcact gtcacagtcg 2820 atcgagtttc tggctatggc cctgggaattttggtatggg aagtcgacta ctggcttcag 2880 gtgaggtacg acacaactca attttatatcatcttcgtgg gcgttttgtt tgccggtcca 2940 agcagcagcc cagaagccga attactccacgagtcttacc aaggctcggt cggctgcgaa 3000 ctatatcctc tggctgcgga cattgaagccgaccatccgc gaaacggagg agaacaagaa 3060 aaaagggcca gtgggtggat gccctgtcgacctcgaggac attgaattca ggtatcgtca 3120 acgtgattcg gctcgagttc tccgcggggtttccatgaca atcgagccag gacaatttgt 3180 agcttatgtg ggcgcttctg gctgtggcaagtcaacgttg atcgctttgt tggaacgatt 3240 ctacgacccg acctcgggcc gaatttcatttgcacacgag aatattgcag aaatgtcgcc 3300 gcgcttgtac cgcggccata tgtctttggtccaacaggaa cccacayttt accaaggctc 3360 cgttcgcgag aatgtgacgt tggccctcgaagccgaatta tcagaagagc tttgtcaagg 3420 acgccttccc gcaaggccaa tgctttggattttgtcatct ctttaccaga aggctttgaa 3480 acgccttgcg gctcaacgag ggatgcagttctccggcggg caacgacagc ggatcgccat 3540 cgcaagagca ttgattcgaa atccaaagctgttgctactt gacgaagcga cgtcagccct 3600 cgacacgcaa tcggaacgtc tggttcaagctgccctcgat gaggcatcca cgagccgaac 3660 gacaatagca gtggcgcacc gactttccactattcggaat gttgatgtta tttttgtgtt 3720 tgccaacggg agaatcgccg aaacgggcactcacgcggaa ctacaacgac tgagaggaag 3780 atattacgag atgtgtttgg cacaatctttagaccaagca tgagcgttca cagagaagcg 3840 gaaaagggcg gtgggatctt ttaggataggtttagtggcg tgttacttac tacaggcgtt 3900 tggattcagg tacgacaact tgtacaataagtagcataga gcatgtaatg aaagggtact 3960 cgtcccggaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaa 3999 10 3792 DNA Exophiala spinifera misc_feature (0)...(0)p-glycoprotein, fully spliced cDNA 10 atggcagatg aatcggagaa acctcgaccaaaccaagatg gcagtgagtc gtcctcacac 60 cctcccccag aaaaggaaac cgaaggcagtatttcagact atctacgaat cttcagatat 120 gccgacaaat acgactggac tctcaatgtcatcgcgctca tctgcgccat cggatccggg 180 gcttcccttc ctctgatgtc gatcatcttcggtagcttca ccaacaagtt caacaattac 240 aattcgggcg acgggagtcc tgaagcgttcaaggccgatg tggatcattt cgtcctgtgg 300 ttcgtctacc tctttattgg gaagtttgtcctcacgtacg tttccacggc tgccattacc 360 atttcagcta tacgaaccac tcgaactcttcgacgagtgt tccttgaatg caccttgcgg 420 caagaggtct ggcatttcga caagcagagcaatggagcaa tcgccactca rgtcactacc 480 aatggcaacc gtatacaaac aggtattgccgagaaattgg tctttaccgt gcaggcactt 540 tcaatgttct tttctgcatt tgtggtcgctttggcgtctc agtggaagct agctttaatc 600 accatgtccg tcatccctgc cattttcctggtcaccggca tctgcatagc aattgatgcc 660 gctcaggagg ccaggatcac caggatctactcacgcgccg ctgtcctcgc agaagaagtc 720 ttatcatcca tccggacagt ccatgctttctacgcccaga agaaaatggt cgaaaaatat 780 gatgtctttt tgcagcaagc acaccaagaagggaagaaga aatcgccaaa taatggsgtc 840 ttgttctcaa ctgagtactt ttgcatttacgctgctatcg cactggcctt ttggaaaggt 900 tttcgcatgt atcagaatgg cgaggttgccgacgttggca aagtctttac tgttgtcctt 960 tccgtcacct tagcagccac gtccatctcaatgcttgcgc cttcaggttc agtcgtttac 1020 caacgccgca tcttcggctc cgaattattcagtatcattg acaaacccac gcagctcgac 1080 cctctcgacc cttctggaaa gcagccagagggctgcctag gtcaaattga gatccaaaac 1140 ctggcatttg cctacccctc ccgaccatctgcccaagtac ttcgagattt caacttgaca 1200 attccagctg gcaagacgac ggccctcgtcggtgcatcag gtagcggcaa aagcacaatg 1260 gtcggcttac ttgaacggtg gtatctgcccagttcgggga ggatattact tgatgggttg 1320 gaactgggac aatacaatgt gaaatggctgagaagccgca ttcgcctcgt tcaacaggaa 1380 cctgtgttgt ttcgtggcac aatcttccagaacattgcca acggtttcat ggatgagcaa 1440 cgagatctgc ctcgcgaaaa acaaatggagcttgtgcaaa aagcttgcaa agccagcaat 1500 ggcgacgtgt tcattaatga gcttccgaacggttatgaga ctgaagttgg cgagcgagcc 1560 ggagccttga gtggaggtca acgacaacgaattgcaatcg cacgaagtat catatcggat 1620 cccaagatcc tgttactcga tgaagctaccagcgcccttg acccgaaggc ggagaaagtg 1680 gtccaggagg ccttgaaccg agtgtccaaagaccgcacta ctttggtcat tgcccacaaa 1740 ctagccactg tcaaaagtgc tggcaacatcgcagtcattt cccaggggaa aatcgtcgag 1800 caaggcacac accacgaatt gatcgaattcggctgtcatt acgccgcact ggtgcgtgca 1860 caggacctcg gggctgacga acaacaagaacatgagaaga ccctgcacga aaaggcagca 1920 cgagaagctg ctggtgaacg accggcacttgagcgcactc acaccactgc cacatctcaa 1980 gctggagacc tggagaagcg gaaggtgccggtcgggactt tgggctactc gctcctaaaa 2040 tgcatcctaa tcatgttcta cgaacaaaaaaatctctact ggtgcttctt gttgtcaaca 2100 ataacggttc tgatatgcgc ggccacatttccaggacaag cccttttgtt ttcgagattg 2160 ctcactgtct tcgagttgag tggtcatgcggcacaggaac gggcagactt ttatattctg 2220 atgttctttg tcgtggctct aggaaatctagtaggatatt tcacgattgg ctggacatgc 2280 aacgttattt cacaagttgt cacccatcgctatcaagccg caatgttcca acgagtactg 2340 gatcaagaca tcgaactcct cgacatcccggagcaaattt ctggtgctct cacatcgcaa 2400 ctgtcagctc tacccacgca gttgcaagagttgatatcag caaattttct catttatatc 2460 gttgtcggtc aacatcgtct cgagcagtgctctaccacta gcctatggat ggaaactggg 2520 cctggtggtt gtgtttggtg cacttccacccctgcttttg gctggctacc tcagaattcg 2580 tctagagacg aagctagaag ccggaaactcggcaaacttt gcagaaagtg ctgggcttgc 2640 aagcgaagca gttaccgcga tccggaccgtctcatctttg actctcgaag gccatgttct 2700 ccaacagtac tcggacatgt tgagcaaggtcttgctaaga tcatccaaag cttttggttt 2760 ggacgatgtt ttggttttca cttgtcacagtcgatggagt ttttggctat tgccctggga 2820 ttttgtattg cagtcgataa ttggcttcaggtgagtacga cacaactcaa ttttatatca 2880 tcttcgtggg cgttttgttt gccggtccaagcagcagccc agtatttggc ttactccacg 2940 agttttacca aggctcggtc ggctgcgaactatatcctct ggctgcggac attgaagccg 3000 accatccgcg aaacggagga gaacaagaaaaaaggcccag tgggtggatg ccctgtcgac 3060 ctcgaggaca ttgaattcag gtatcgtcaacgtgattcgg ctcgagttct ccgcggggtt 3120 tccatgacaa tcgagccagg acaatttgtagcttatgtgg gcgcttctgg ctgtggcaag 3180 tcaacgttga tcgctttgtc ggaacgattctacgacccga cctcgggccg aatttcattt 3240 gcacacgaga atattgcaga aatgtcgccgcgcttgtacc gcggccatat gtctttggtc 3300 caacaggaac ccacacttta ccaaggctccgttcgcgaga atgtgacgtt ggccctcgaa 3360 gccgaattat cagaagagct ttgtcaaggacgccttcccg caaggccaat gctttggatt 3420 ttgtcatctc tttaccagaa ggctttgaaacgccttgcgg ctcaacgagg gatgcagttc 3480 tccggcgggc aacgacagcg gatcgccatcgcaagagcat tgattcgaaa tccaaagctg 3540 ttgctacttg acgaagcgac gtcagccctcgacacgcaat cggaacgtct ggttcaagct 3600 gccctcgatg aggcatccac gagccgaacgacaatagcag tggcgcaccg actttccact 3660 attcggaatg ttgatgttat ttttgtgtttgccaacggga gaatcgccga aacgggcact 3720 cacgcggaac tacaacgact gagaggaagatattacgaga tgtgtttggc acaatcttta 3780 gaccaagcat ga 3792 11 1263 PRTExophiala spinifera VARIANT 157 Xaa = Any Amino Acid 11 Met Ala Asp GluSer Glu Lys Pro Arg Pro Asn Gln Asp Gly Ser Glu 1 5 10 15 Ser Ser SerHis Pro Pro Pro Glu Lys Glu Thr Glu Gly Ser Ile Ser 20 25 30 Asp Tyr LeuArg Ile Phe Arg Tyr Ala Asp Lys Tyr Asp Trp Thr Leu 35 40 45 Asn Val IleAla Leu Ile Cys Ala Ile Gly Ser Gly Ala Ser Leu Pro 50 55 60 Leu Met SerIle Ile Phe Gly Ser Phe Thr Asn Lys Phe Asn Asn Tyr 65 70 75 80 Asn SerGly Asp Gly Ser Pro Glu Ala Phe Lys Ala Asp Val Asp His 85 90 95 Phe ValLeu Trp Phe Val Tyr Leu Phe Ile Gly Lys Phe Val Leu Thr 100 105 110 TyrVal Ser Thr Ala Ala Ile Thr Ile Ser Ala Ile Arg Thr Thr Arg 115 120 125Thr Leu Arg Arg Val Phe Leu Glu Cys Thr Leu Arg Gln Glu Val Trp 130 135140 His Phe Asp Lys Gln Ser Asn Gly Ala Ile Ala Thr Xaa Val Thr Thr 145150 155 160 Asn Gly Asn Arg Ile Gln Thr Gly Ile Ala Glu Lys Leu Val PheThr 165 170 175 Val Gln Ala Leu Ser Met Phe Phe Ser Ala Phe Val Val AlaLeu Ala 180 185 190 Ser Gln Trp Lys Leu Ala Leu Ile Thr Met Ser Val IlePro Ala Ile 195 200 205 Phe Leu Val Thr Gly Ile Cys Ile Ala Ile Asp AlaAla Gln Glu Ala 210 215 220 Arg Ile Thr Arg Ile Tyr Ser Arg Ala Ala ValLeu Ala Glu Glu Val 225 230 235 240 Leu Ser Ser Ile Arg Thr Val His AlaPhe Tyr Ala Gln Lys Lys Met 245 250 255 Val Glu Lys Tyr Asp Val Phe LeuGln Gln Ala His Gln Glu Gly Lys 260 265 270 Lys Lys Ser Pro Asn Asn GlyVal Leu Phe Ser Thr Glu Tyr Phe Cys 275 280 285 Ile Tyr Ala Ala Ile AlaLeu Ala Phe Trp Lys Gly Phe Arg Met Tyr 290 295 300 Gln Asn Gly Glu ValAla Asp Val Gly Lys Val Phe Thr Val Val Leu 305 310 315 320 Ser Val ThrLeu Ala Ala Thr Ser Ile Ser Met Leu Ala Pro Ser Gly 325 330 335 Ser ValVal Tyr Gln Arg Arg Ile Phe Gly Ser Glu Leu Phe Ser Ile 340 345 350 IleAsp Lys Pro Thr Gln Leu Asp Pro Leu Asp Pro Ser Gly Lys Gln 355 360 365Pro Glu Gly Cys Leu Gly Gln Ile Glu Ile Gln Asn Leu Ala Phe Ala 370 375380 Tyr Pro Ser Arg Pro Ser Ala Gln Val Leu Arg Asp Phe Asn Leu Thr 385390 395 400 Ile Pro Ala Gly Lys Thr Thr Ala Leu Val Gly Ala Ser Gly SerGly 405 410 415 Lys Ser Thr Met Val Gly Leu Leu Glu Arg Trp Tyr Leu ProSer Ser 420 425 430 Gly Arg Ile Leu Leu Asp Gly Leu Glu Leu Gly Gln TyrAsn Val Lys 435 440 445 Trp Leu Arg Ser Arg Ile Arg Leu Val Gln Gln GluPro Val Leu Phe 450 455 460 Arg Gly Thr Ile Phe Gln Asn Ile Ala Asn GlyPhe Met Asp Glu Gln 465 470 475 480 Arg Asp Leu Pro Arg Glu Lys Gln MetGlu Leu Val Gln Lys Ala Cys 485 490 495 Lys Ala Ser Asn Gly Asp Val PheIle Asn Glu Leu Pro Asn Gly Tyr 500 505 510 Glu Thr Glu Val Gly Glu ArgAla Gly Ala Leu Ser Gly Gly Gln Arg 515 520 525 Gln Arg Ile Ala Ile AlaArg Ser Ile Ile Ser Asp Pro Lys Ile Leu 530 535 540 Leu Leu Asp Glu AlaThr Ser Ala Leu Asp Pro Lys Ala Glu Lys Val 545 550 555 560 Val Gln GluAla Leu Asn Arg Val Ser Lys Asp Arg Thr Thr Leu Val 565 570 575 Ile AlaHis Lys Leu Ala Thr Val Lys Ser Ala Gly Asn Ile Ala Val 580 585 590 IleSer Gln Gly Lys Ile Val Glu Gln Gly Thr His His Glu Leu Ile 595 600 605Glu Phe Gly Cys His Tyr Ala Ala Leu Val Arg Ala Gln Asp Leu Gly 610 615620 Ala Asp Glu Gln Gln Glu His Glu Lys Thr Leu His Glu Lys Ala Ala 625630 635 640 Arg Glu Ala Ala Gly Glu Arg Pro Ala Leu Glu Arg Thr His ThrThr 645 650 655 Ala Thr Ser Gln Ala Gly Asp Leu Glu Lys Arg Lys Val ProVal Gly 660 665 670 Thr Leu Gly Tyr Ser Leu Leu Lys Cys Ile Leu Ile MetPhe Tyr Glu 675 680 685 Gln Lys Asn Leu Tyr Trp Cys Phe Leu Leu Ser ThrIle Thr Val Leu 690 695 700 Ile Cys Ala Ala Thr Phe Pro Gly Gln Ala LeuLeu Phe Ser Arg Leu 705 710 715 720 Leu Thr Val Phe Glu Leu Ser Gly HisAla Ala Gln Glu Arg Ala Asp 725 730 735 Phe Tyr Ile Leu Met Phe Phe ValVal Ala Leu Gly Asn Leu Val Gly 740 745 750 Tyr Phe Thr Ile Gly Trp ThrCys Asn Val Ile Ser Gln Val Val Thr 755 760 765 His Arg Tyr Gln Ala AlaMet Phe Gln Arg Val Leu Asp Gln Asp Ile 770 775 780 Glu Leu Leu Asp IlePro Glu Gln Ile Ser Gly Ala Leu Thr Ser Gln 785 790 795 800 Leu Ser AlaLeu Pro Thr Gln Leu Gln Glu Leu Ile Ser Ala Asn Phe 805 810 815 Leu IleTyr Ile Val Val Gly Gln His Arg Leu Glu Gln Cys Ser Thr 820 825 830 ThrSer Leu Trp Met Glu Thr Gly Pro Gly Gly Cys Val Trp Cys Thr 835 840 845Ser Thr Pro Ala Phe Gly Trp Leu Pro Gln Asn Ser Ser Arg Asp Glu 850 855860 Ala Arg Ser Arg Lys Leu Gly Lys Leu Cys Arg Lys Cys Trp Ala Cys 865870 875 880 Lys Arg Ser Ser Tyr Arg Asp Pro Asp Arg Leu Ile Phe Asp SerArg 885 890 895 Arg Pro Cys Ser Pro Thr Val Leu Gly His Val Glu Gln GlyLeu Ala 900 905 910 Lys Ile Ile Gln Ser Phe Trp Phe Gly Arg Cys Phe GlyPhe His Leu 915 920 925 Ser Gln Ser Met Glu Phe Leu Ala Ile Ala Leu GlyPhe Cys Ile Ala 930 935 940 Val Asp Asn Trp Leu Gln Val Ser Thr Thr GlnLeu Asn Phe Ile Ser 945 950 955 960 Ser Ser Trp Ala Phe Cys Leu Pro ValGln Ala Ala Ala Gln Tyr Leu 965 970 975 Ala Tyr Ser Thr Ser Phe Thr LysAla Arg Ser Ala Ala Asn Tyr Ile 980 985 990 Leu Trp Leu Arg Thr Leu LysPro Thr Ile Arg Glu Thr Glu Glu Asn 995 1000 1005 Lys Lys Lys Gly ProVal Gly Gly Cys Pro Val Asp Leu Glu Asp Ile 1010 1015 1020 Glu Phe ArgTyr Arg Gln Arg Asp Ser Ala Arg Val Leu Arg Gly Val 1025 1030 1035 1040Ser Met Thr Ile Glu Pro Gly Gln Phe Val Ala Tyr Val Gly Ala Ser 10451050 1055 Gly Cys Gly Lys Ser Thr Leu Ile Ala Leu Ser Glu Arg Phe TyrAsp 1060 1065 1070 Pro Thr Ser Gly Arg Ile Ser Phe Ala His Glu Asn IleAla Glu Met 1075 1080 1085 Ser Pro Arg Leu Tyr Arg Gly His Met Ser LeuVal Gln Gln Glu Pro 1090 1095 1100 Thr Leu Tyr Gln Gly Ser Val Arg GluAsn Val Thr Leu Ala Leu Glu 1105 1110 1115 1120 Ala Glu Leu Ser Glu GluLeu Cys Gln Gly Arg Leu Pro Ala Arg Pro 1125 1130 1135 Met Leu Trp IleLeu Ser Ser Leu Tyr Gln Lys Ala Leu Lys Arg Leu 1140 1145 1150 Ala AlaGln Arg Gly Met Gln Phe Ser Gly Gly Gln Arg Gln Arg Ile 1155 1160 1165Ala Ile Ala Arg Ala Leu Ile Arg Asn Pro Lys Leu Leu Leu Leu Asp 11701175 1180 Glu Ala Thr Ser Ala Leu Asp Thr Gln Ser Glu Arg Leu Val GlnAla 1185 1190 1195 1200 Ala Leu Asp Glu Ala Ser Thr Ser Arg Thr Thr IleAla Val Ala His 1205 1210 1215 Arg Leu Ser Thr Ile Arg Asn Val Asp ValIle Phe Val Phe Ala Asn 1220 1225 1230 Gly Arg Ile Ala Glu Thr Gly ThrHis Ala Glu Leu Gln Arg Leu Arg 1235 1240 1245 Gly Arg Tyr Tyr Glu MetCys Leu Ala Gln Ser Leu Asp Gln Ala 1250 1255 1260

That which is claimed:
 1. An isolated polypeptide comprising the aminoacid sequence set forth in SEQ ID NO:3.
 2. An isolated polypeptidecomprising an amino acid sequence having at least 70% identity to thesequence of SEQ ID NO:3, wherein said polypeptide retains monooxygenaseactivity.
 3. An isolated polypeptide comprising a fragment of SEQ IDNO:3, wherein said fragment retains monooxygenase activity.
 4. Thepolypeptide of claim 1, wherein said polypeptide is encoded by thenucleotide sequence of SEQ ID NO:2.
 5. The isolated polypeptide of claim3, wherein said fragment comprises at least 100 contiguous amino acidsof SEQ ID NO:3 and retains monooxygenase activity.
 6. The polypeptide ofclaim 2, wherein said polypeptide comprises an amino acid sequencehaving at least 95% identity to the sequence of SEQ ID NO:3, whereinsaid polypeptide retains monooxygenase activity.